Further studies are clearly needed to determine whether the PI3K/Akt signaling pathway is usually involved in the protective effect of VP. In summary, VP by acting upon V1 receptors protects neuronal cells from serum deprivation-induced apoptosis. C (PKC). Western blot analyses exposed time-dependent decreases of Bad phosphorylation and raises in cytosolic levels of cytochrome c following serum deprivation, effects which were prevented by 10nM VP. These data demonstrate that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. The data suggest that VP offers anti-apoptotic activity in neurons and that VP may act as a neuroprotective agent in the brain. for 5 min, washed twice with ice-cold PBS and resuspended in 0.3 ml of PBS containing 2% FBS. Forward Scatter (FSC) and Part Scatter (SSC) of cells were acquired by a FACSCalibur circulation cytometer (Becton Dickinson, CA) and analyzed by FlowJo software (TreeStar, San Jose, CA). FSC shows cell size, and SSC is related to cell granularity or internal difficulty. Living cells were gated based on cell optic characteristics (FSC and SSC). FACS detection of apoptotic cells The degree of apoptosis following serum deprivation and VP treatment was examined by FACS, based on the ability of fluorescence-labeled annexin V to bind phosphatidyl serine, which is definitely translocated to the outer membrane coating during early apoptosis, and the capacity of amino-actinomycin D to bind to the nuclei of late apoptotic cells. H32 cells (2.5 105) were incubated with 5ul of annexin V-FITC (1 mg/ml) and 7-Amino-actinomycin D (7-AAD) (1 mg/ml) (BD Biosciences) for quarter-hour at room heat, according to the manufacturers instructions, and immediately analyzed by circulation cytometry as described above. This method allows discrimination of early apoptotic cells (annexin V+ /7-AAD?) and late apoptotic cells (annexin V+ /7-AAD+) (17). Early apoptotic cells (annexin V+ /7-AAD?) and late apoptotic cells (annexin V+/7-AAD+) were counted for total apoptosis. Caspase-3 activity measurement Caspase-3 activity was measured using a Caspase-3/CPP32 fluorometric protease assay kit (BioSource International, Inc., Camarillo, CA) according to the manufacturer’s protocol. Briefly, cells were washed with PBS, centrifuged for 5 min at 800 for 10 min at 4 C, and protein concentrations in the supernatants identified using BCA? protein Assay (PIERCE, Rockford, IL). Aliquots comprising 100g of protein were incubated with substrate DEVD (Asp-Glue-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) for 90 min at 37 C. Upon cleavage of the substrate by Caspase-3, free AFC, which emits a yellow-green fluorescence, was measured by using a FLUOStar OPTIMA microplate reader (BMG Labtechnologies Inc, Durham, NC), having a 405 nm excitation and 505 nm emission filter. Cytosolic cytochrome c levels The levels of cytosolic cytochrome were measured using a Cytochrome c ELISA Kit (MBL, Watertown, MA). Briefly, H32 cells were cultured in 100mm tradition flasks, serum deprived for 0, 0.5, 1, 2, 4 and 6h in the absence or in the presence of VP (10 nM). After treatment, the cells were harvested using trypsin-EDTA, spun down at 200 for 5 min, washed twice with ice-cold PBS and resuspended in 500 L ice-cold homogenization buffer (10 mm Tris/HCl (pH 7.5), 0.3 m sucrose, 25 g/mL aprotinin, 1 mm phenylmethylsulphonyl fluoride, and 10 g/mL leupeptin). Cells were then homogenized on snow using a dounce homogenizer and centrifuged at 10 000 for 60 min at 4 C. Protein concentrations in the supernatants (cytosolic fractions) were identified using BCA? protein Assay (Pierce, Rockford, IL). Cytosolic cytochrome level was recognized using peroxidase conjuagted anti-cytochrome c polyclonal antibody, according to the manufacturer’s instructions. Western blot analysis Western blot analysis was performed essentially as explained previously (18). Briefly, cells were lysed with T-PER Cells Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with proteinase and phosphate inhibitor cocktail (Sigma). Protein concentrations were determined by BCA? Protein Assay (Pierce) and 20 g of protein were loaded and separated inside a 4C20% SDS-PAGE (Invitrogen,). Proteins were transferred from your gel to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ), incubated with 5% nonfat dried milk in Tri-buffered saline (TBS plus 0.1% Tween-20 (TBST)) for 1h and incubated with the antibodies at a 1:1,000 dilution overnight. After washing JDTic in TBST, membranes were incubated for 2h with peroxidase-linked anti-Rabbit IgG at a 1:10,000 dilution or anti-mouse IgG at a 1:5,000. -tubulin was used to correct for protein loading. Detection of immunoreactive band was performed by using ECL Plus TM reagents (Amersham Pharmacia Biotech) and contact with BioMax MR film (Kodak, Rochester, NY). Densitometric quantification from the immunoblots was performed utilizing the open public domain NIH Picture.[PubMed] [Google Scholar] 8. deprivation-induced apoptosis, through phosphorylation-inactivation from the pro-apoptotic proteins, Poor, and consequent reduces in cytosolic cytochome c and caspase-3 activation. The info claim that VP provides anti-apoptotic activity in neurons which VP may become a neuroprotective agent in the mind. for 5 min, cleaned double with ice-cold PBS and resuspended in 0.3 ml of PBS containing 2% FBS. Forwards Scatter (FSC) and Aspect Scatter (SSC) of cells had been acquired with a FACSCalibur movement cytometer (Becton Dickinson, CA) and examined by FlowJo software program (TreeStar, San Jose, CA). FSC signifies cell size, and SSC relates to cell granularity or inner intricacy. Living cells had been gated predicated on cell optic features (FSC and SSC). FACS recognition of apoptotic cells The amount of apoptosis pursuing serum deprivation and VP treatment was analyzed by FACS, predicated on the power of fluorescence-labeled annexin V to bind phosphatidyl serine, which is certainly translocated towards the external membrane level during early apoptosis, and the capability of amino-actinomycin D to bind towards the nuclei lately apoptotic cells. H32 cells (2.5 105) had been incubated with 5ul of annexin V-FITC (1 mg/ml) and 7-Amino-actinomycin D (7-AAD) (1 mg/ml) (BD Biosciences) for a quarter-hour at room temperatures, based on the producers guidelines, and immediately analyzed by movement cytometry as described above. This technique enables discrimination of early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+ /7-AAD+) (17). Early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+/7-AAD+) had been counted for total apoptosis. Caspase-3 activity dimension Caspase-3 activity was assessed utilizing a Caspase-3/CPP32 fluorometric protease assay package (BioSource International, Inc., Camarillo, CA) based on the manufacturer’s process. Briefly, cells had been cleaned with PBS, centrifuged for 5 min at 800 for 10 min at 4 C, and proteins concentrations in the supernatants motivated using BCA? proteins Assay (PIERCE, Rockford, IL). Aliquots formulated with 100g of proteins had been incubated with substrate DEVD (Asp-Glue-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) for 90 min at 37 C. Upon cleavage from the substrate by Caspase-3, free of charge AFC, which emits a yellow-green fluorescence, was assessed with a FLUOStar OPTIMA microplate audience (BMG Labtechnologies Inc, Durham, NC), using a 405 nm excitation and 505 nm emission filtration system. Cytosolic cytochrome c amounts The degrees of cytosolic cytochrome had been measured utilizing a Cytochrome c ELISA Package (MBL, Watertown, MA). Quickly, H32 cells had been cultured in 100mm lifestyle flasks, serum deprived for 0, 0.5, 1, 2, 4 and 6h in the absence or in the current presence of VP (10 nM). After treatment, the cells had been gathered using trypsin-EDTA, spun down at 200 for 5 min, cleaned double with ice-cold PBS and resuspended in 500 L ice-cold homogenization buffer (10 mm Tris/HCl (pH 7.5), 0.3 m sucrose, 25 g/mL aprotinin, 1 mm phenylmethylsulphonyl fluoride, and 10 g/mL leupeptin). Cells had been after that homogenized on glaciers utilizing a dounce homogenizer and centrifuged at 10 000 for 60 min at 4 C. Proteins concentrations in the supernatants (cytosolic fractions) had been motivated using BCA? proteins Assay (Pierce, Rockford, IL). Cytosolic cytochrome level was discovered using peroxidase conjuagted anti-cytochrome c polyclonal antibody, based on the manufacturer’s guidelines. Western blot evaluation Western blot evaluation was performed essentially as referred to previously (18). Quickly, cells had been lysed with T-PER Tissues Proteins Removal Reagent (Pierce, Rockford, IL) supplemented with proteinase and phosphate inhibitor cocktail (Sigma). Proteins concentrations had been dependant on BCA? Proteins Assay (Pierce) and 20 g of proteins had been packed and separated within a 4C20% SDS-PAGE (Invitrogen,). Protein had been transferred through the gel to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ), incubated with 5% non-fat dried dairy in Tri-buffered saline (TBS plus 0.1% Tween-20 (TBST)) for 1h and incubated using the antibodies at a 1:1,000 dilution overnight. After cleaning in TBST, membranes had been incubated for 2h with peroxidase-linked anti-Rabbit IgG at a 1:10,000 dilution or anti-mouse IgG at a 1:5,000. -tubulin was utilized to improve for proteins loading. Recognition of immunoreactive music group was performed through the use of ECL Plus TM reagents (Amersham Pharmacia Biotech) and contact with BioMax MR film (Kodak, Rochester, NY). Densitometric quantification from the immunoblots was performed utilizing the open public domain NIH Picture plan (ImageJ 1.36b developed in the US Country wide Institutes of Wellness, and on the Internet in: Rabbit polyclonal to Kinesin1 http://rsb.Info.nih.gov/nih-image). Data evaluation Statistical need for the distinctions between groups.Traditional western blot analysis using antibody against phospho-Bad(Ser112), revealed time-dependent decreases in phospho-Bad levels, with significant reduction by 2 h and a tendency for an additional decrease up to 6 h (Fig. of cytochrome c pursuing serum deprivation, results which were avoided by 10nM VP. These data show that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation from the pro-apoptotic proteins, Poor, and consequent reduces in cytosolic cytochome c and caspase-3 activation. The info claim that VP offers anti-apoptotic activity in neurons which VP may become a neuroprotective agent in the mind. for 5 min, cleaned double with ice-cold PBS and resuspended in 0.3 ml of PBS containing 2% FBS. Forwards Scatter (FSC) and Part Scatter (SSC) of cells had been acquired with a FACSCalibur movement cytometer (Becton Dickinson, CA) and examined by FlowJo software program (TreeStar, San Jose, CA). JDTic FSC shows cell size, and SSC relates to cell granularity or inner difficulty. Living cells had been gated predicated on cell optic features (FSC and SSC). FACS recognition of apoptotic cells The amount of apoptosis pursuing serum deprivation and VP treatment was analyzed by FACS, predicated on the power of fluorescence-labeled annexin V to bind phosphatidyl serine, which can be translocated towards the external membrane coating during early apoptosis, and the capability of amino-actinomycin D to bind towards the nuclei lately apoptotic cells. H32 cells (2.5 105) had been incubated with 5ul of annexin V-FITC (1 mg/ml) and 7-Amino-actinomycin D (7-AAD) (1 mg/ml) (BD Biosciences) for quarter-hour at room temp, based on the producers guidelines, and immediately analyzed by movement cytometry as described above. This technique enables discrimination of early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+ /7-AAD+) (17). JDTic Early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+/7-AAD+) had been counted for total apoptosis. Caspase-3 activity dimension Caspase-3 activity was assessed utilizing a Caspase-3/CPP32 fluorometric protease assay package (BioSource International, Inc., Camarillo, CA) based on the manufacturer’s process. Briefly, cells had been cleaned with PBS, centrifuged for 5 min at 800 for 10 min at 4 C, and proteins concentrations in the supernatants established using BCA? proteins Assay (PIERCE, Rockford, IL). Aliquots including 100g of proteins had been incubated with substrate DEVD (Asp-Glue-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) for 90 min at 37 C. Upon cleavage from the substrate by Caspase-3, free of charge AFC, which emits a yellow-green fluorescence, was assessed with a FLUOStar OPTIMA microplate audience (BMG Labtechnologies Inc, Durham, NC), having a 405 nm excitation and 505 nm emission filtration system. Cytosolic cytochrome c amounts The degrees of cytosolic cytochrome had been measured utilizing a Cytochrome c ELISA Package (MBL, Watertown, MA). Quickly, H32 cells had been cultured in 100mm tradition flasks, serum deprived for 0, 0.5, 1, 2, 4 and 6h in the absence or in the current presence of VP (10 nM). After treatment, the cells had been gathered using trypsin-EDTA, spun down at 200 for 5 min, cleaned double with ice-cold PBS and resuspended in 500 L ice-cold homogenization buffer (10 mm Tris/HCl (pH 7.5), 0.3 m sucrose, 25 g/mL aprotinin, 1 mm phenylmethylsulphonyl fluoride, and 10 g/mL leupeptin). Cells had been after that homogenized on snow utilizing a dounce homogenizer and centrifuged at 10 000 for 60 min at 4 C. Proteins concentrations in the supernatants (cytosolic fractions) had been established using BCA? proteins Assay (Pierce, Rockford, IL). Cytosolic cytochrome level was recognized using peroxidase conjuagted anti-cytochrome c polyclonal antibody, based on the manufacturer’s guidelines. Western blot evaluation Western blot evaluation was performed essentially as referred to previously (18). Quickly, cells had been lysed with T-PER Cells Proteins Removal Reagent (Pierce, Rockford, IL) supplemented with proteinase and phosphate inhibitor cocktail (Sigma). Proteins concentrations had been dependant on BCA? Proteins Assay (Pierce) and 20 g of proteins had been packed and separated inside a 4C20% SDS-PAGE (Invitrogen,). Protein had been transferred through the gel to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ), incubated with 5% non-fat dried dairy in Tri-buffered saline (TBS plus 0.1% Tween-20 (TBST)) for 1h and incubated using the antibodies at a 1:1,000 dilution overnight. After cleaning in TBST, membranes had been incubated for 2h with peroxidase-linked anti-Rabbit IgG at a 1:10,000 dilution or anti-mouse IgG at a 1:5,000. -tubulin was utilized to improve for proteins loading. Recognition of immunoreactive music group was performed through the use of ECL Plus TM reagents (Amersham Pharmacia Biotech) and contact with BioMax MR film (Kodak, Rochester, NY). Densitometric quantification from the immunoblots was performed utilizing the general public domain NIH Picture system (ImageJ 1.36b developed in the US Country wide Institutes of Wellness, and.313:9C12. cleaned double with ice-cold PBS and resuspended in 0.3 ml of PBS containing 2% FBS. Forwards Scatter (FSC) and Part Scatter (SSC) of cells had been acquired with a FACSCalibur movement cytometer (Becton Dickinson, CA) and examined by FlowJo software program (TreeStar, San Jose, CA). FSC shows cell size, and SSC relates to cell granularity or inner difficulty. Living cells had been gated predicated on cell optic features (FSC and SSC). FACS recognition of apoptotic cells The amount of apoptosis pursuing serum deprivation and VP treatment was analyzed by FACS, predicated on the power of fluorescence-labeled annexin V to bind phosphatidyl serine, which can be translocated towards the external membrane coating during early apoptosis, and the capability of amino-actinomycin D to bind towards the nuclei lately apoptotic cells. H32 cells (2.5 105) had been incubated with 5ul of annexin V-FITC (1 mg/ml) and 7-Amino-actinomycin D (7-AAD) (1 mg/ml) (BD Biosciences) for quarter-hour at room temp, based on the producers guidelines, and immediately analyzed by movement cytometry as described above. This technique enables discrimination of early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+ /7-AAD+) (17). Early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+/7-AAD+) had been counted for total apoptosis. Caspase-3 activity dimension Caspase-3 activity was assessed utilizing a Caspase-3/CPP32 fluorometric protease assay package (BioSource International, Inc., Camarillo, CA) based on the manufacturer’s process. Briefly, cells had been cleaned with PBS, centrifuged for 5 min at 800 for 10 min at 4 C, and proteins concentrations in the supernatants established using BCA? proteins Assay (PIERCE, Rockford, IL). Aliquots including 100g of proteins had been incubated with substrate DEVD (Asp-Glue-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) for 90 min at 37 C. Upon cleavage from the substrate by Caspase-3, free of charge AFC, which emits a yellow-green fluorescence, was assessed with a FLUOStar OPTIMA microplate audience (BMG Labtechnologies Inc, Durham, NC), having a 405 nm excitation and 505 nm emission filtration system. Cytosolic cytochrome c amounts The degrees of cytosolic cytochrome had been measured utilizing a Cytochrome c ELISA Package (MBL, Watertown, MA). Quickly, H32 cells had been cultured in 100mm tradition flasks, serum deprived for 0, 0.5, 1, 2, 4 and 6h in the absence or in the current presence of VP (10 nM). After treatment, the cells had been gathered using trypsin-EDTA, spun down at 200 for 5 min, cleaned double with ice-cold PBS and resuspended in 500 L ice-cold homogenization buffer (10 mm Tris/HCl (pH 7.5), 0.3 m sucrose, 25 g/mL aprotinin, 1 mm phenylmethylsulphonyl fluoride, and 10 g/mL leupeptin). Cells had been after that homogenized on glaciers utilizing a dounce homogenizer and centrifuged at 10 000 for 60 min at 4 C. Proteins concentrations in the supernatants (cytosolic fractions) had been driven using BCA? proteins Assay (Pierce, Rockford, IL). Cytosolic cytochrome level was discovered using peroxidase conjuagted anti-cytochrome c polyclonal antibody, based on the manufacturer’s guidelines. Western blot evaluation Western blot evaluation was performed essentially as defined previously (18). Quickly, cells had been lysed with T-PER Tissues Proteins Removal Reagent (Pierce, Rockford, IL) supplemented with proteinase and phosphate inhibitor cocktail (Sigma). Proteins concentrations had been dependant on BCA? Proteins Assay (Pierce) and 20 g of proteins had been packed and separated within a 4C20% SDS-PAGE (Invitrogen,). Protein had been transferred in the gel to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ), incubated with 5% non-fat dried dairy in Tri-buffered saline (TBS plus 0.1% Tween-20 (TBST)) for 1h and incubated using the antibodies at a 1:1,000 dilution overnight. After cleaning in TBST, membranes had been incubated for 2h with peroxidase-linked anti-Rabbit IgG at.The bars represent the mean S.E.M of three tests conducted in duplicate. cytosolic degrees of cytochrome c pursuing serum deprivation, results which were avoided by 10nM VP. These data show that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation from the pro-apoptotic proteins, Poor, and consequent reduces in cytosolic cytochome c and caspase-3 activation. The info claim that VP provides anti-apoptotic activity in neurons which VP may become a neuroprotective agent in the mind. for 5 min, cleaned double with ice-cold PBS and resuspended in 0.3 ml of PBS containing 2% FBS. Forwards Scatter (FSC) and Aspect Scatter (SSC) of cells had been acquired with a FACSCalibur stream cytometer (Becton Dickinson, CA) and examined by JDTic FlowJo software program (TreeStar, San Jose, CA). FSC signifies cell size, and SSC relates to cell granularity or inner intricacy. Living cells had been gated predicated on cell optic features (FSC and SSC). FACS recognition of apoptotic cells The amount of apoptosis pursuing serum deprivation and VP treatment was analyzed by FACS, predicated on the power of fluorescence-labeled annexin V to bind phosphatidyl serine, which is normally translocated towards the external membrane level during early apoptosis, and the capability of amino-actinomycin D to bind towards the nuclei lately apoptotic cells. H32 cells (2.5 105) had been incubated with 5ul of annexin V-FITC (1 mg/ml) and 7-Amino-actinomycin D (7-AAD) (1 mg/ml) (BD Biosciences) for a quarter-hour at room heat range, based on the producers guidelines, and immediately analyzed by stream cytometry as described above. This technique enables discrimination of early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+ /7-AAD+) (17). Early apoptotic cells (annexin V+ /7-AAD?) and past due apoptotic cells (annexin V+/7-AAD+) had been counted for total apoptosis. Caspase-3 activity dimension Caspase-3 activity was assessed utilizing a Caspase-3/CPP32 fluorometric protease assay package (BioSource International, Inc., Camarillo, CA) based on the manufacturer’s process. Briefly, cells had been cleaned with PBS, centrifuged for 5 min at 800 for 10 min at 4 C, and proteins concentrations in the supernatants driven using BCA? proteins Assay (PIERCE, Rockford, IL). Aliquots filled with 100g of proteins had been incubated with substrate DEVD (Asp-Glue-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) for 90 min at 37 C. Upon cleavage from the substrate by Caspase-3, free of charge AFC, which emits a yellow-green fluorescence, was assessed with a FLUOStar OPTIMA microplate audience (BMG Labtechnologies Inc, Durham, NC), using a 405 nm excitation and 505 nm emission filtration system. Cytosolic cytochrome c amounts The degrees of cytosolic cytochrome had been measured utilizing a Cytochrome c ELISA Package (MBL, Watertown, MA). Quickly, H32 cells had been cultured in 100mm lifestyle flasks, serum deprived for 0, 0.5, 1, 2, 4 and 6h in the absence or in the current presence of VP (10 nM). After treatment, the cells had been gathered using trypsin-EDTA, spun down at 200 for 5 min, cleaned double with ice-cold PBS and resuspended in 500 L ice-cold homogenization buffer (10 mm Tris/HCl (pH 7.5), 0.3 m sucrose, 25 g/mL aprotinin, 1 mm phenylmethylsulphonyl fluoride, and 10 g/mL leupeptin). Cells had been after that homogenized on ice using a dounce homogenizer and centrifuged at 10 000 for 60 min at 4 C. Protein concentrations in the supernatants (cytosolic fractions) were decided using BCA? protein Assay (Pierce, Rockford, IL). Cytosolic cytochrome level was detected using peroxidase conjuagted anti-cytochrome c polyclonal antibody, according to the manufacturer’s instructions. Western blot analysis Western blot analysis was performed essentially as explained previously (18). Briefly, cells were lysed with T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with proteinase and phosphate inhibitor cocktail (Sigma). Protein concentrations were determined by BCA? Protein Assay (Pierce) and 20 g of protein were loaded and separated in a 4C20% SDS-PAGE (Invitrogen,). Proteins were transferred from your gel to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ), incubated with 5% nonfat dried milk in Tri-buffered saline (TBS plus 0.1% Tween-20 (TBST)) for 1h and incubated with the antibodies at a 1:1,000 dilution overnight. After washing in TBST, membranes were incubated for 2h with peroxidase-linked anti-Rabbit IgG at a 1:10,000 dilution or anti-mouse IgG at a 1:5,000. -tubulin was used to correct for protein loading. Detection of immunoreactive band was performed by using ECL Plus TM reagents (Amersham Pharmacia Biotech) and exposure to BioMax MR film (Kodak, Rochester, NY). Densitometric quantification of the immunoblots was performed by using the public domain NIH Image program (ImageJ 1.36b developed at the US National Institutes of Health, and available on the Internet at: http://rsb.Info.nih.gov/nih-image). Data analysis Statistical significance of the differences between groups was calculated by one-way analysis of variance.