Cell lysis buffer

Tested cells were lysed using the cell lysis buffer (Cwbio, Beijing, China) containing 1% phenylmethylsulfonyl fluoride, and the protein content of the lysates was quantified using the BCA protein assay kit (Cwbio). Cell protein extraction was evaluated by performing Western blot analysis as previously described [21 (link)], with primary antibodies including Anti-Hsp90α (Proteintech, Rosemont, IL, USA), Anti-Hsp27, Hsp70, protein kinase B (Akt), phosphorylated Akt (p-Akt), M2 isoform of pyruvate kinase (PKM2), Heat shock factor -1 (HSF-1), and Cytochrome C (Cyt-C, ZENBIO, Chengdu, Sichuan, China). HRP-conjugated second antibodies (Abbkine, Wuhan, Hubei, China) were used. Finally, reactive bands were quantified using the Quantity One software (ver. 4.6.2; Bio-Rad Laboratories, Hercules, CA, USA). β-actin was used as the loading control.

Protein preparation—Total cellular protein was isolated with cell lysis buffer (CWBIO, Beijing China), and membrane and cytoplasmic protein fractions of cultured cells were obtained with a Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific).
Immunoprecipitation—Flag fusion proteins were precipitated with anti-Flag-conjugated beads (Sigma, St. Louis, MO, USA). The proteins bound to beads were eluted with SDS sample buffer and analyzed by SDS-PAGE and quantitative Western blotting.
Immunoblots—Protein samples were loaded on an 8% or 10% polyacrylamide SDS gel and transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). The membranes were blocked in Tris-buffered saline (TBS; 20 mM Tris-Cl, pH 7.6, 137 mM NaCl) with 5% skim milk. Blots were incubated with the primary antibody diluted in TBS + 5% milk overnight at 4 C, washed three times in TBS + 0.5% Tween 20, and then incubated for 45 min in the peroxidase-conjugated secondary antibody. Blots were developed using the ECL detection kit.
The antibodies used in this study were as follows: anti-Filamin A (Abcam, Rabbit, 1:250,000), anti-Filamin A (phospho S2152) (Abcam, Rabbit, 1:10,000, Cambridge, UK); anti-Caveolin-1 (Proteintech, Rabbit, 1:5000, Wuhan, China); anti-mCherry (TDY bio, Mouse, 1:5000, Beijing, China); anti-Beta-tubulin (TDY bio, Mouse, 1:5000, Beijing, China).