Hrp linked anti rabbit igg antibody

HEK-293 T cells were seeded into 6-well plates and 5 × 10⁵ HEK-293 T cells per well transfected with siRhoA (200 nM) or RhoA over-expression plasmids (4 m/mL), or their controls; 48 h after incubation, the cells were treated with IFN-a (1000 U/mL) for an additional 6 h or the RhoA/ROCK inhibitor Y27632 (60 μM, Beyotime), which was added for an additional 45 min before adding IFN-a. The cells then were harvested and lysed at different time points and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for immunoblotting followed by protein detection with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). The visualized proteins were scanned and the signal intensities quantified using Image J. The specified primary antibodies were directed against RhoA (Santa Cruz Biotechnology, diluted 1:200), total and phosphorylated STAT1 and STAT2, (Proteintech, diluted 1:3000), and β-actin (Abcam, diluted 1:5000). The secondary antibodies were horseradish-peroxidase (HRP)-linked anti-mouse IgG antibody (Proteintech 1:10,000) and HRP-linked anti-rabbit IgG antibody (Proteintech, diluted 1:10,000).

HEK-293T cells were seeded into 6-well plates and 5×10⁵ HEK-293T cells per well transfected with siRhoA (200 nM) or RhoA over-expression plasmids (4 μ/mL), or their controls; 48 hours after incubation, the cells were treated with IFN-a (1,000 units/mL) for an additional 6 hours or RhoA/ROCK inhibitor Y27632 (60 μM, Beyotime ) was added for an additional 45 minutes before adding IFN-a. The cells then were harvested and lysed at different time points and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for immunoblotting followed by protein detection with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). The visualized proteins were scanned and signal intensities quantified using Image J. The specified primary antibodies were directed against RhoA (Santa Cruz Biotechnology, diluted 1:200), and total and phosphorylated STAT1 and STAT2, (Proteintech, diluted 1:3000), and β-actin (Abcam, diluted 1:5000). The secondary antibodies were horseradish-peroxidase (HRP)-linked anti-mouse IgG antibody (Proteintech 1:0000), and HRP-linked anti-rabbit IgG antibody (Proteintech, diluted 1:10000).