Anti vinculin v4139

Western blot analysis was performed as previously described (Ibuki et al., 2014 (link); Saneyasu et al., 2015 ). Anti-p-S6 (S235/236) (#2211), anti-Akt (#9272), anti-p-Akt (S473) (#9271), and horseradish peroxidase-conjugated anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Beverly, MA, USA). As a loading control, anti-vinculin (V4139) was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). After the detection of bands, membranes were rinsed with Restore™ Plus Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA), and used for reprobing with proper antibodies.

After T_reg differentiation, whole-cell extracts have been lysed in radioimmunoprecipitation assay and centrifuged. Cells were then sonicated, and the entire lysates were loaded. Anti-LC3B (L7543) and anti-vinculin (v4139) were purchased from Sigma-Aldrich. Anti-p62 (MBL-M162-3) was purchased from MBL. Opa1 (ab157457) was purchased from Abcam. AMPK substrate antibody sampler kit (#35277) was purchased from Cell Signaling Technology. PINK1 (ab23707) was purchased from Abcam. Parkin-Ser⁶⁵ (PA5-114616) was purchased from Thermo Fisher Scientific. The Odyssey system (LI-COR) was used for detection, and the ImageJ software was used for quantification. Vinculin has been used as a loading control. To detect OXPHOS complex assembly, we used an assembly-dependent total OXPHOS rodent antibody cocktail (Abcam, ab110413). The antibodies in the cocktail are against a subunit that is labile when its complex is not assembled. The samples were prepared following the manufacturer’s protocol.