Supersignal west pico plus solution

Cells were lysed on ice using RIPA buffer (Thermo Fisher Scientific) with a cocktail of proteinase inhibitors. Protein lysates were electrophoresed on 10% SDS polyacrylamide gels before transfer to PVDF membranes (MilliporeSigma, Burlington, MA). Membranes were blocked with 5% skimmed milk and incubated with primary antibodies: anti-ACSL4 (Abcam, ab155282), anti-RAB22A (Proteintech, 12125-1-AP), anti-SERINC3 (LSBio, LS-C386356), or anti-GAPDH (GeneTex, GTX100118) overnight at 4 °C. Subsequently, goat anti-rabbit HRP antibodies (1:5000, Bio-Rad) were applied for 1 h at room temperature. After washing with 1% TBST, membranes were developed with enhanced chemiluminescence (SuperSignal West Pico PLUS) solution (Thermo Fisher Scientific) and exposed to a ChemiDoc imaging system (Bio-Rad, Hercules, CA). Data were analyzed using ImageJ software (NIH).

Cell pellets were prepared with lysis buffer (50mM Tris pH 7.6, 2% SDS) with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentrations were measured using Pierce BCA assay (Thermo Scientific). Separated proteins were transferred on the PVDF membrane. The membranes were blocked with 1% nonfat milk in TBST for 1h at room temperature. After blocking, the membranes were incubated overnight at 4°C with primary antibodies listed in Supplementary table 5. Bands were developed using SuperSignal West Pico Plus Solution (Thermo Scientific) and detected with an autoradiography CL-Xposure Film (Thermo Scientific). Films were scanned and images were quantified with Image J software.

Whole-cell lysates were extracted with lysis buffer (50 mM Tris pH 7.6, 2% SDS) with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentrations were measured using Pierce BCA assay (Thermo Scientific). Separated proteins were transferred to the PVDF membrane. The membranes were blocked with 1% nonfat milk in TBST for 1 h at room temperature. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies listed in Supplementary Table 5. Bands were developed using SuperSignal West Pico Plus Solution (Thermo Scientific) and detected with an autoradiography CL-Xposure Film (Thermo Scientific). Films were scanned, and images were quantified with Image J software.