Phospho pdk1 ser241

Cells extracts were prepared and analyzed as previously described [16 (link)]. Western blotting was performed by anti-ZKSCAN1 (Bioss, P17029), FBXW7 Rabbit Polyclonal Antibody (ABclonal, A5872), mTOR Mouse Monoclonal antibody (Proteintech, 66,888–1-lg), β-Actin Rabbit mAb (ABclonal, AC026), anti-His Mouse Monoclonal antibody (ABclonal, AE003), HA-tag antibody (ABclonal, AE008), anti DDDDK-Tag Mouse Monoclonal antibody (ABclonal, AE005), anti-GAPDH (Sangon Biotech, D110016), AKT Polyclonal Antibody (Proteintech, 51,077–1-AP), Phospho-Akt (Ser473) (Cell Signaling Technology, 4060), Phospho-Akt (Thr308) (Cell Signaling Technology, 13,038), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, 4370), ERK1/2 Monoclonal Antibody (67170–1-Ig), Phospho-mTOR (Ser2448) (Cell Signaling Technology, 5536), Phospho-PDK1 (Ser241) (Cell Signaling Technology, 3438), Phospho-MDM2 (Ser166) (Cell Signaling Technology, 3521), Phospho-p70 (S6) Kinase (Thr389) (Cell Signaling Technology, 97,596), MDM2 (Proteintech, 66,511–1-Ig), PDK1 (Proteintech, 18,262–1-AP), P70 (S6K) (Proteintech, 14,485–1-AP), Biotinylated Rabbit anti-Goat IgG (ZSGB-BIO, ZB-2050) and Biotinylated Mouse anti-Goat IgG (ZSGB-BIO, ZB-2020) antibodies. The antigen-antibody complexes were visualized by chemiluminescence.

Appropriate cells were lyzed in RIPA lysis buffer containing a cocktail of protease inhibitors (Roche) and PMSF. Total protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Ding Guo Biotechnology, Shanghai, China). Antibodies against the following proteins were used for immunoblotting: phospho-Akt (Thr308) (#2965S , dilutions 1:1000), phospho-Akt (Ser473) (#4060S, dilutions 1:1000), Akt (#4691L, dilutions 1:1000), phospho-PDK1 (Ser241) (#3438s, dilutions 1:1000), and PDK1 (#3062, dilutions 1:1000), phospho-GSK-3β (Ser9) (#9323P, dilutions 1:1000), GSK-3β (#9315L, dilutions 1:1000), β-catenin (#8480, dilutions 1:1000), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370S, dilutions 1:1000), p44/42 MAPK (Erk1/2) (#4695S, dilutions 1:1000), cleaved caspase 3 (#9315L, dilutions 1:1000), all from Cell Signaling Technology (MA, USA). PARP1 (#13371-1-AP, dilutions 1:1000), PPP1R15A (#10449-1-AP, dilutions 1:500), (ProteinTech, Rosemont, IL, USA); PPP1α (#271762, dilation 1:1000) and β-actin (#47778, dilutions 1:2000), (Santa Cruz Biotechnology, Santa Cruz, CA, USA); YY1 (#61779, dilutions 1:500, Active Motif, CA, USA). The immunoblots were scanned using an Odyssey infrared imaging system (LI-COR). Immunolabeling was detected using the ECL reagent (Sigma). Protein expression was normalized against β-actin.

The following antibodies and reagents were purchased: CDK4 (#12790), CDK6 (#13331), cyclin D1 (#55506), cyclin E (#20808), p21 (#2947), p27 (#3686), PARP (#9532), caspase-9 (#9504), cleaved-caspase-3 (#9664), AKT (#4691), phospho-AKT (Ser473) (#4060), phospho-PDK1 (Ser241) (#3438), PTEN (#9188), phospho-PTEN (Ser380) (#9551) and phospho-c-raf (Ser259) (#9421) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Bcl-2 (ab32124), Bcl-XL (ab32370), Bax (ab32503) and cytochrome c (ab133504) antibodies were purchased from abcam. GAPDH (60004-1-Ig), Survivin (10508-1-AP), XIAP (66800-1-Ig), COXIV (11242-1-AP) and PCNA (60097-1-Ig) antibodies were purchased from Proteintech Group (Chicago, IL, USA). Rhein (R7269), Oxaliplatin (O9512) and N-acetyl-L-cysteine (NAC, A7250) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DCFH-DA (S0033) and JC-1 (C2006) were purchased from Beyotime (Haimen, China). Z-DEVD-FMK (S7312), z-LEHD-FMK (S7313) and z-VAD-FMK (S8102) were purchased from Selleck (Houston, TX, USA). LY294002 (HY-10108) and 740Y-P (HY-P0175) were purchased from MCE (Monmouth, NJ, USA).

Compound 9za (HPLC purity: 96.9%) was synthesized by Central South University. The Annexin V-FITC/PI Apoptosis Kit, Cell Cycle Detection Kit, Colorimetric TUNEL System, Mitochondrial Membrane Potential Assay Kit with JC-1 and MTT were purchased from MultiSciences (Hangzhou, China), KeyGEN (Nanjing, China), Promega (Madison, USA), Beyotime (Shanghai, China) and Sigma-Aldrich (St. Louis, Missouri, USA), respectively. Antibodies against active caspase-3, active caspase-9, Akt, phospho-Akt (Ser473), BAX, Bcl-2, CDK4, CDK6, Cyclin D1, Cytochrome C, PARP were purchased from Abcam (Cambridge, UK). Antibodies to detect the protein levels of ERK1/2, phospho-ERK1/2 (Thy202/Tyr204), MEK1/2, phospho-MEK1/2 (Ser217/221) and phospho-PDK1 (Ser241) were obtained from Cell Signaling Technology (Boston, USA). PD0325901 and BX517 were obtained from MedChemExpress (Shanghai, China).

Cell lysates after various treatments were prepared and subjected to IB analysis as described (Gu et al., 2007 (link)). The sources of primary antibodies were as follows: Cullin-1 (Santa Cruz, CA), phospho-AKT-Ser473, phosphor-AKT-Thr308, pan-AKT, AKT1, AKT2, AKT3, Aurora A, VHL, Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11), PDK1, phospho-PDK1-Ser²⁴¹ and Rictor (53A2) (Cell Signaling, Denver, CO), γ-tubulin, α-tubulin Clone AA13, acetylated tubulin Clone 6-11B-1, β-actin (Sigma, St. Louis, MO), CP110, NDE1, Cep97, Arl13B (Proteintech, Wuhan, P.R.C). UBA3 (Abcam, Shanghai, P.R.C). EGFR and phospho-EGFR-Tyr845 were gifts from Dr. Wanhong Xu,