Subcellular proteome extraction kit

Cell pellets were suspended in 0.1 ml of ice-cold RIPA buffer and incubated on ice for 1 h. When subcellular fractions were prepared, the Subcellular Proteome Extraction Kit (Merck, Darmstadt, Germany) was used according to the manufacturer’s instructions. Protein concentrations were determined by DC protein assay (Bio-Rad, Hercules, California, USA) and equivalent amounts of total cellular protein were separated by 3–8% or 4–12% gradient gels. The proteins were electrophoresed using a polyacrylamide gel and the resolved proteins were transferred to a polyvinylidene fluoride membrane and detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) after a specific antibody reaction. Anti-β-actin and anti-Mcl-1 antibodies were purchased from Sigma-Aldrich and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively. Anti-p-IkB, anti-NF-κB, anti-Bcl-2, anti-Bcl-xL, anti-p-p38, anti-cleaved caspase-3, and anti-Lamin A/C antibodies were purchased from Cell Signaling Technology. Results were analyzed and quantified using a CS Analizer 3 (Atto Corporation, Tokyo, Japan).

WB analysis was performed as previously described [22 (link)]. For the total lysate, cells were lysed in lysis buffer containing 1% of NP40, 250 mM of Tris, 150 mM of NaCl, 0.25% of protease inhibitor, and 1% of phosphatase inhibitor. The lysates were centrifuged, boiled with SDS containing buffer for 10 min for protein denaturation and unfolding, and stored at −20 °C freezer until usage. For the detection of proteins in different cell compartments, lysate was first fractionated with a subcellular proteome extraction kit (Millipore, Burlington, MA, USA, #539790), and then denatured in a similar way for analysis. For the Western blotting (The uncropped blots from this paper’s Figures are shown in Supplementary File S1), an equal amount of protein samples was added to SDS-polyacrylamide gel for resolving. The resolved protein samples were then transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membrane was then blocked by 5% of non-fat milk. After 1 h, the primary antibodies (listed in Table 1) against target proteins were applied and then incubated for 2 h, followed by washing with 5% TBST. Subsequently, HRP-conjugated secondary antibody incubation was conducted, and substrates were added for the detection of the protein. The protein bands were detected by exposure to X-ray films.