Ab207450

Cells for protein extraction were grown in T75 flasks, washed with cold PBS, scraped, pelleted and frozen in −80 °C, before they were lysed using RIPA lysis buffer. 20 µg protein was loaded into 6–12% SDS-PAGE gels from Bio-Rad Laboratories (Hercules, CA, USA). Blotting was performed by using the Bio Rad Trans-Blot Turbo system and blocking was performed for 2 h in 5% milk in TBS-T (0.1% (v/v) Tween). Primary antibodies used were PDK1 (ab207450, Abcam, Cambridge, UK), and p-PDH (ab92696, Abcam), E-Cadherin (CDH1, 14472S, cell signaling), vimentin (VIM, ab92547, Abcam), LDHA (3582, Cell signaling). The housekeeping protein a-tubulin or vinculin was used as loading control when total protein staining was not available. Primary antibodies were removed, and the membranes were washed three times for 5 min with TBS-T before incubating them with secondary antibody for 1 h under agitation. The membranes were washed three times with TBS-T before they were examined. Complete blots can be found in Supplementary Figure S4.

Cells were lysed in Laemmli buffer (0.125 M Tris-HCl, pH 6.8, 2% SDS, 5% β-Mercaptoethanol, 10% glycerine, 0.002% bromophenol blue and 1% protease inhibitor cocktail (APExBIO, K1007)). The lysates were separated on 10% or 12% (wt/vol) SDS-polyacrylamide gels. After SDS/PAGE, proteins were transferred to PVDF membrane for Western blotting in transfer buffer with 10% (vol/vol) methanol at 4°C followed by standard Western-blotting protocols. β-actin or α-Tubulin was used for normalizing the protein load. All blots were repeated at least three times. Comparisons of relative levels of specific antigens were done by quantitative densitometry using ImageJ Software (version 1.48). Antibodies: HIF1α (Cell Signaling Technology, 36169), BNIP3 (Cell Signaling Technology, 3769), Ubiquitin (Cell Signaling Technology, 3936), P70S6K (Cell Signaling Technology, 2708), p-P70S6K (Cell Signaling Technology, 9234), PDK1 (Abcam, ab207450), mTOR (Abcam, ab134903), p-mTOR (Abcam, ab109268), AKT1/2/3 (Abcam, ab179463), p-AKT1/2/3 (Abcam, ab192623), LC3II (Zen BioScience, 306019), β-actin (Zen BioScience, 700068), α-Tubulin (Beyotime, AF0001), peroxidase-conjugated anti-rabbit (Beyotime, A0216) and peroxidase-conjugated anti-mouse antibodies (Beyotime, A0216).

The cells were lysed with radio immunoprecipitation assay (RIPA) peptide lysis buffer (R0010, Beijing Solarbio Science and Technology Co. Ltd., Beijing, China) to extract the total protein. The proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with rat anti-human primary antibodies PTBP1 (ab133734, 1: 1000, Abcam Inc., Cambridge, UK), GLUT3 (ab41525, 1: 1000, Abcam), HK2 (ab209847, 1: 1000, Abcam), and PDK1 (ab207450, 1: 1000, Abcam) at 4° C overnight. Next, the membrane was washed three times with TBST (5 min per time), followed by incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (1: 20000; ab205718, Abcam) for 1 h. Protein blots were visualized by ECL-associated fluorography (Merck Millipore, Billerica, MA, USA). The data was analyzed using ImageJ 1.48u (National Institutes of Health Inc., USA). The protein content was reflected by the ratio of gray value between proteins and the internal reference (GAPDH).