Western Blot Analysis of PRDX6 Protein

Protein samples were separated by electrophoresis on 12% SDS-PAGE gels and transferred to PVDF membranes (GE Healthcare). The western blot procedure was performed as described previously (Shanmugasundaram et al., 2017 (link)) with minor modifications. Briefly, membranes were blocked for 1 h in 5% non-fat milk, 1× TBS, 0.1% Tween-20 and probed with the corresponding primary and secondary antibodies. For detection of PRDX6 protein the membrane was incubated in 5% non-fat milk, 1× TBS, 0.1% Tween-20 with 1:1,000 polyclonal rabbit anti-PRDX6 antibody (ab59543, Abcam) 1.5 h at room temperature followed by incubation for 1 h at room temperature with 1:15,000 hP-conjugated anti-rabbit secondary antibody (ab6721, Abcam). Beta-actin as a loading control was detected by incubation in 5% non-fat dry milk, 1× TBS, 0.1% Tween-20 with 1:10,000 anti-β-Actin antibody (4967S, Cell Signalling) 1.5 h at room temperature followed by incubation for 1 h at room temperature in 1:20,000 anti-rabbit polyclonal antibody (ab6721, Abcam). The signals were quantified using ImageJ 1.52a (NIH) software based on the calculation of peak area and the intensity values for PRDX6 bands were normalized across the samples by the actin control. Statistical significance of differences between the group means was evaluated by ANOVA with Tukey correction for multiple comparisons in Prism 6 (GraphPad Software).