Western Blot Protein Quantification

Samples were homogenized in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific. Cat. 23225) or Bradford Assay (Cat. ab119216, Abcam). The blot was performed using 15 to 20 µg of protein according to previously described methods [26 (link), 27 (link)]. Primary and secondary antibodies used are detailed in Tables 1 and 2. Membranes were visualized with enhanced chemiluminescence (Clarity Western ECL Blotting Substrates; Bio-Rad Laboratories) and bands detected using Syngene G-Box imaging system (Syngene). Western Blot analyses were performed using ImageJ software and densitometry normalized to loading control GAPDH or Rab11.