Western Blot Analysis of Protein Samples

Cells were lysed in Triton Lysis Buffer (150 mM NaCl, 25 mM Tris pH 7,4, 1% Triton X-100 supplemented with cOmplete EDTA-free protease inhibitor cocktail) or RIPA (150 mM NaCl, 50 mM Tris pH 8, 1% NP40, 0.5% NaDoc, 0.1% SDS, supplemented with cOmplete EDTA-free protease inhibitor cocktail) as indicated. Cell lysates were denatured in 4X LDS/100 mM DTT and resolved on 4 to 12% Bris-Tris or 3 to 8% Tris Acetate SDS-PAGE gels under reducing conditions and transferred to a polyvinylidene difluoride membrane (PVDF, 0.45 μM, Thermo Fisher Scientific) or Millipore Immobilon FL for near-infrared fluorescence detection by LI-COR Clx. Membranes were blocked in 5% (w/v) nonfat dried skimmed milk powder in PBST (PBS, 0.1% Tween-20) for 1 h at room temperature (RT). Membranes were then probed with the appropriate primary antibodies in blocking buffer overnight at 4 °C. Detection was performed by incubating membranes with the appropriate horseradish peroxidase (HRP)-conjugated or IRDye secondary antibodies in blocking buffer at RT for 1 h. Enhanced chemiluminescence (ECL) (Thermo Fisher Scientific) was used for anti-ubiquitin western blots, and images were acquired on a FUSION-SL imager (Vilber Lourmat, France). Alternatively, anti-GST blots were visualized on a LI-COR Clx (46 (link)).