Western Blotting Protocol for Protein Analysis

Western blotting was performed as described previously [52 (link)]. Briefly, cells were lysed in SDS sample buffer containing 2 µg/mL aprotinin (Seikagaku Kogyo, Tokyo, Japan), 0.8 µg/mL pepstain A (Wako Pure Chemicals, Osaka, Japan), 2 µg/mL leupeptin (Nacalai Tesque), 2 mM PMSF (Nacalai Tesque), 20 mM β-glycerophosphate (MilliporeSigma), 50 mM NaF (Wako), and 10 mM Na₃VO₄ (Wako) and denatured at 40 and 100 °C for 20 and 5 min, respectively. Cell lysate components were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with Blocking One (Nacalai Tesque) and incubated for 1 h at room temperature or overnight at 4 °C with primary and secondary antibodies diluted in Tris-buffered saline containing 5% Blocking One and 0.1% Tween20. Sequential reprobing of the membranes with various antibodies was performed after the inactivation of HRP by 0.1% NaN₃. Proteins were detected with Chemi-Lumi One L (07880-70, Nacalai Tesque) and Clarity (#1705061, Bio-Rad, Hercules, CA, USA) using an image analyzer ChemiDoc XRSplus (Bio-Rad). Full-length blots of Figure 1, Figure 3, Figure 6, and Figure S2 are shown in Figure S5–S8.