Bicinchoninic acid assay

Whole-cell protein lysate preparation was performed with RIPA buffer (15 mM HEPES, 150 mM NaCl, 10 mM EGTA, 2% Triton X-100) supplemented with protease and phosphatase inhibitors (complete mini EDTA-free and PhosSTOP, Roche) as described before [14 (link),21 (link),35 ]. Bicinchoninic acid assay (Santa Cruz Biotechnology, Texas, USA) was used for protein quantitation according to the manufacturer's protocol. Laemmli buffer was mixed with cell lysates, denatured for 5 min at 95 °C and loaded in a 10% polyacrylamide gel (Thermo Fisher Scientific). Lysates were separated by electrophoresis at 120 V and transferred to a PVDF membrane. Membranes were blocked in 5% nonfat dry milk in TBS-T and incubated in primary antibody in 5% nonfat dry milk in TBS-T overnight at 4 °C. Antibodies and dilutions are listed in Supplementary Table 1. Membranes were washed three times and incubated with the secondary antibody. Proteins were visualized with ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and the Fusion FX7 imaging system was used for detection (Vilber Lourmat).

Total proteins were extracted using a protein extraction kit (ProMab Biotechnologies, Richmond, CA, USA). Lysates were centrifugated at 12,000 rpm for 5 min at 4°C. Total protein level in the supernatant was quantified using bicinchoninic acid assay (Santa Cruz Biotechnology, USA). Subsequently, samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto equilibrated polyvinylidene fluoride membrane (Millipore, Bradford, MA, USA). The membrane was blocked with 5% non-fat dried milk in Tris-buffered saline-Tween 20 solution and incubated with rabbit anti-FSIP1 antibody at a dilution of 1:200 (Abcam, Cambridge, UK) at 4°C overnight. After thorough washing, membranes were incubated with HRP-conjugated anti-rabbit secondary antibody at a dilution of 1:1,000 (Cell Signaling Technology, Danvers, MA, USA) at 37°C for 2 h. The bands of targeted proteins were visualized on the membrane using a Pierce enhanced chemiluminescence system kit (Thermo Fisher Scientific, Rockford, IL, USA) and quantitatively analyzed using a DNR BioImaging System (DNR, Jerusalem, Israel). β-actin served as a loading control for normalization.