Ab52944

For western blot analysis, total proteins were extracted using the Whole Cell Protein Extraction Kit (Key GEN, China). A BCA Protein Quantitation Assay (Thermo, USA) was used to measure the protein concentration. We separated protein samples by using 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gels. The separated protein samples were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% nonfat dry milk in Tris-buffered saline (TBS)/0.1% Tween 20 for 1 h at room temperature, the membranes were incubated with AKT (1:1000, 10176-2-AP, Proteintech), PI3K (1:1000, 60225-1-Ig, Proteintech), p-PI3K (1:1000, 28731-1-AP, Proteintech), p-PI3K (1:1000, ab182651, Abcam), PGK1 (1:1000, 17811-1-AP, Proteintech), Ser82 VIMENTIN (1:1000, ab52943-10, Abcam), Ser72 VIMENTIN (1:1000, ab52944, Abcam), Ser38 VIMENTIN (1:1000, ab52942, Abcam), Ser83 VIMENTIN (1:1000, 3878 T, CST, USA) and anti-GAPDH (1:1000, 5,174, CST, USA). The next day, membranes were washed 3 times with TBST buffer for 15 min and incubated with horseradish peroxidase-conjugated secondary antibody. Finally, the western blot signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA).

Primary antibodies used for Western blot, immunohistochemistry and immunocytochemistry are listed as followed: Hif-1α (79233, Cell signaling, USA), α-SMA (ab32575, Abcam, USA), Bnip3 (ab10433, Abcam, USA; ab38621, Abcam, USA), LC3B (83506, cell signaling, USA; 12741, cell signaling, USA), vimentin (ServiceBio, GB12192, China), phosphor-vimentin Ser56 (ab217673, Abcam, USA), phosphor-vimentin Ser38 (ab52942, Abcam, USA), phosphor-vimentin Ser72 (ab52944, Abcam, USA), phosphor-vimentin Ser82 (ab52943, Abcam, USA), caspase 3 (ab197202, Abcam, USA), cytochrome C (sc-13156, Santa Cruz, USA), GAPDH (GB12002, Servicebio, China).