Western Blotting Protocol for Protein Analysis

Western blotting was performed as previously described (Hu et al., 2015 (link)). In brief, proteins were extracted and separated by 10% (w/v) SDS-PAGE, then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, United States). The membrane was blocked in 5% skimmed milk, and then incubated with primary antibody overnight at 4°C. After washing with TBST (10 mM Tris. HCl, pH 7.6, 150 mM NaCl, Tween 20 0.1%), the membranes were incubated with Goat anti-Rabbit IgG Secondary Antibody, HRP (31460, ThermoFisher, United States) or Goat anti-Mouse IgG Secondary Antibody, HRP (31430, ThermoFisher, United States) for 1 h and washed twice with TBST. The membranes were developed using the ECL (Enhanced chemiluminescence) kit (K-12045-D50, Advansta, United States), visualized, and recorded with the use of an Imagequant LAS 4000 mini machine (GE Healthcare Life Sciences, United States). Primary antibodies: Plzf (sc-28319, Santa Cruz Biotechnology, United States), Stra8 (ab49405, Abcam, United States), Scp3 (ab97672, Abcam, United States), Ptbp2 (ab154787, Abcam, United States), and ubiquitin (#3936, CST, United States).