Anti phospho pdk1

Western blot analysis was performed as described previously [33 (link)] with minor modifications. Briefly, aliquots of 30–60 µg of protein were subjected to 8–12% SDS-PAGE and transferred onto Hybond-P⁺ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with the following primary antibodies: anti-ICAM-1 (ab225884, 1:1000, Abcam, Cambridge, UK), anti-VCAM-1 (ab106778, 1:1000, Abcam), anti-phospho-ERK (sc-7383, 1:1000, Santa Cruz Biotechnology), anti-total-ERK (sc-94 1:1000, Santa Cruz Biotechnology), anti-phospho-PKC (9375S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PKC (sc-10800, 1:1000, Santa Cruz Biotechnology), anti-phospho-PDK1 (3061S, 1:1000, Cell Signaling Technology), anti-PDK1 (3062S, 1:1000, Cell Signaling Technology), anti-phospho-STAT-3 (9131S, 1:1000, Cell Signaling Technology, anti-STAT-3 (4904S, 1:1000, Cell Signaling Technology), anti-HIF-1α (ab2185, 1:1000, Abcam), anti-phospho-NF-κB (8242S, 1:1000, Cell Signaling Technology), anti-NF-κB (8242S 1:1000, Cell Signaling Technology), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich, St. Louis, MO, USA). The bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and an ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA).

Cholesteatoma keratinocytes were cultured on 35 mm culture dishes. The cells were grown to 70-80% confluence and then placed in KSFM with control (DMSO), GW0742 (100 nM), or GSK0660 (5 μM). After 24 h of treatment, the cells were washed and isolated using cell lysis buffer (Beyotime Institute of Biotechnology, China) containing protease inhibitors. Equal amounts of total protein were separated on 8% SDS-PAGE and transferred to a PVDF membrane (100 V for 60 min). The membrane was incubated with the primary antibodies overnight at 4°C, followed by the secondary peroxidase-conjugated antibody for 1 h. The bands were visualized by enhanced chemiluminescence and exposure to ECL Hyperfilm (GE Healthcare). The densitometry of bands was quantified with NIH Image 1.63 software. The protein expression was normalized to the amount of beta-actin. The following primary antibodies were used: anti-phospho-PDK1, anti-protein kinase B (AKT), anti-phospho-AKT, anti-phospho-GSK3β, and anti-phospho-PTEN (all from Cell Signaling Technology, Danvers, MA, USA). Antibodies against β-actin, Cyclin D1, and PPAR β/δ were from Genetex, Inc. (Genetex, CA, USA).

The following reagents and materials were used: anti-GAPDH (Calbiochem), Anti-OXSR1, anti-NONO (Abcam), anti-Prohibitin-1 (cell signaling), anti-dimethylated histone H3 (Lys9) (Upstate), anti-p38 MAP kinase, anti-phospho p38 MAP kinase (Thr180/Tyr 182), anti-PDK1, anti-phospho PDK1 (S241), anti-PKC-Pan, anti-phospho PKC-pan (T514), anti-pAkt (Ser473), anti-Akt, anti-pERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-STAT3, anti-phospho STAT3 (Y705), anti-cJUN, anti-phospho cJUN (S73), anti-NF-κB p65, anti-NF-κB phospho-p65 (S536), anti-PLCgamma, anti-phospho PLCgamma (Y783), anti-SAPK/JNK, and anti-phospho SAPK/JNK (T183/Y185) (Cell Signaling). Electrophoresis reagents were purchased from Bio-rad and trypsin from Promega.