Anti pdpk1

Proteins were isolated from cells using M-PER (Thermo Scientific, MA, USA) separated in Mini-PROTEAN TGX Gel (4–12%, Bio-Rad) and electrotransferred onto a PVDF membrane (Millipore). After blocking in Blocking One (Nacalai Tesque, Kyoto, Japan), the membranes were incubated for 1 h at room temperature with primary antibodies, which included anti-CD63 (purified mouse anti-human CD63, H5C6, 1:200, BD), anti-CD9 (ALB6, 1:200, Santa Cruz Biotechnology Inc.), anti-cytochrome C (purified mouse anti-cytochrome C, 7H8.2C12, 1:200, BD), anti-Claudin-5 (Z43.JK, 1:200, Invitrogen), anti-Occludin (ZMD.481, 1:200, Invitrogen), anti-ZO-1 (H-300, 1:100, Santa Cruz Biotechnology), anti-N-cadherin (3B9, 1:500, Invitrogen), anti-PDPK1 (#3062, 1:500, Cell Signaling), anti-GAPDH (6C5, 1:1000, Millipore), anti-Cofilin (D3F9, 1:1000, Cell Signaling), anti-Phospho-Cofilin (Ser3) (#3311, 1:500, Cell Signaling) and anti-N-SMase2 (H-195, 1:200, Santa Cruz Biotechnology Inc.). Secondary antibodies (HRP-linked anti-mouse IgG, NA931 or HRP-linked anti-rabbit IgG, NA934, GE Healthcare) were used at a dilution of 1:2,000. The membrane was then exposed to ImmunoStar LD (Wako, Osaka, Japan). Original scans of the cropped images in the figures are presented in Supplementary Figs 7–13.

Anti-ERp57 (ab10287) and Anti-pAkt (Thr308) (ab194875) antibodies were from Abcam (Cambridge, UK). Anti-β-Actin (A2103) antibody, PDPK1 inhibitor GSK2334470 (SML0217), Nocodazole (M1404) and human Insulin (91077C) were from Sigma-Aldrich (Munich, Germany). Anti-p53 (GTX102965) antibody was from GeneTex (Hsinchu City, Taiwan). Anti-PUMA (ABC158) antibody was from Millipore (Burlington, MA, USA). Anti-HIF1α (610,959) was from BD Transduction Laboratories (San Jose, CA, USA). Anti-c-Myc (#13,987), Anti-ERK1/2 (#4695), Anti-GFP (#2956), Anti-HA-Tag (#3724), Anti-pAkt (Ser473) (#4060), Anti-p–c-Myc (#13,748), Anti-pERK1/2 (#4370), Anti-pGSK3β (#5558), Anti-PDPK1 (#3062), Anti-PLK1(#4513), Anti-p-p70S6K (#9205), Anti-pPDPK1 (#3438), Anti-pPLK1 (#9062), Anti-PCNA (#2586), Anti-V5-Tag (#13,202) antibodies and PI3K inhibitor LY294002 (#9901) were obtained from Cell Signaling Technology. DMOG (BML-EI347) was purchased from Enzo Life Sciences (Lörrach, Germany).