Phospho pdk1

Resveratrol, anti-β-actin antibody, and the c-Myb inhibitor, celastrol, were purchased from Sigma-Aldrich. For Western blotting, monoclonal antibodies against phospho-STAT5, STAT5, phospho-Akt, Akt, phospho-Erk, Erk, phospho-PDK1, PDK1, phospho-rictor, rictor, and c-Myb were purchased from Cell Signaling Technology (Danvers, MA, USA).

Electrophoresis SDS-PAGE (Laemmli) was used. The protein was transferred to 0.2 μm pore-sized PVDF membrane (GE Healthcare, Amersham, UK), either at 150 mA or 100 V for 1 h by using a Bio-Rad Mini Trans- Blot electrophoresis cell. The membrane was incubated in a 1:2500 dilution of monoclonal mouse anti-human phospho-PTEN (Ser380), AKT (pan), phospho -AKT (T308), phospho-GSK-3-beta (Ser9), phospho-PDK1 (Ser241), phospho-c-Raf (Ser259), m-TOR, phospho-mTOR (Ser2448), phospho-p70 S6 (Ser371) and phospho-4E-BP1 (Thr37/46) (Cell Signaling, Beverly, MA, USA) at 4 °C overnight.
PVDF samples were incubated in Amersham ECL Western blotting detection analysis system (Amersham, UK). The results were standardized using the beta-actin expression in a sample and were expressed in percentages to the protein content in non-transformed tissues. The level of protein in normal kidney parenchyma was indicated as 100%.

The following primary antibodies were used in this study: deTyr-Tubulin (rabbit, G.G.Gundersen, Columbia University, New York, USA), total Tubulin (mouse, clone B5-1-2, Sigma), Tubulin tyrosine ligase (rabbit, ProteinTech), PTEN (rabbit, clone 138G6, Cell Signaling), Tau1 (mouse, clone PC1C6, Chemicon), phospho-FAK (Y397) (rabbit, clone D20B1, Cell Signaling), total FAK (rabbit, clone D2R2E, cell signaling), phospho-PDK1, (rabbit, clone C49H2, Cell Signaling), phospho-Akt (Ser473), (rabbit, clone D9E, Cell Signaling), total Akt (rabbit, clone C67E7, cell signaling), phospho-GSK3β, (rabbit, cell signaling), total GSK3β (mouse, clone 7/GSK-3b, BD transduction), β-Actin (mouse, clone AC-15, Sigma), GAPDH (mouse, clone 71,1, Sigma), GFP (chicken, abcam), acytelated Tubulin (mouse, clone 6-11B-1, Sigma), tyrosinated Tubulin (rat, clone YL1/2, abcam) and GM130 (rabbit, clone EP892Y, abcam). Secondary antibodies for immunofluorescence labeled with Alexa Fluor dyes (488, 568 and 647) were purchased from Invitrogen. HRP-coupled antibodies for immunoblotting were from Dianova/Jackson ImmunoResearch.

Puerarin (HY-N0145) and PTEN inhibitor (VO-Ohpic, HY-13074) were purchased from MEC (USA); miR-21 mimic was purchased from RIBOBIO (miR10000530-1-5, China). The following antibodies were purchased from Cell Signaling Technology (USA): E-cadherin (3195, 1:1,000), N-cadherin (13116, 1:1,000), Vimentin (5741, 1:1,000), Snail (3879, 1:1,000), Slug (9585, 2:1,000), PTEN (7960, 1:1,000), PDK1 (3062, 1:1,000), phospho-PDK1 (Ser241) (3438, 1:1,000), phospho-Akt (Ser473) (4060, 1:1,000), AKT (4691, 1:1,000), GSK-3β (12456, 1:1,000), phospho-GSK-3β (Ser9)(5558, 1:1,000), Ki67 (9027, 1:400), and GAPDH (5174, 1:1,000). All reagents and antibodies were used according to the manufacturer's instructions.

Western blots were conducted following previously described methods [30 (link)]. The rabbit polyclonal antibodies against ALDOB (Proteintech, Rosemont, IL, USA, Cat. 18065-1-AP, diluted 1:5000), LDHA (Proteintech, Cat. 21799-1-AP, diluted 1:1000), and LDHB (Proteintech, Cat. 14824-1-AP, diluted 1:5000), along with the rabbit monoclonal antibody against ACTB (Sigma-Aldrich, St. Louis, MO, USA, Cat. A5441, diluted 1:10000), PDK1 (Cell Signaling Technology, Danvers, MA, USA, Cat. #5662, diluted 1:1000), phospho-PDK1 (Cell Signaling Technology, Cat. #3438, diluted 1:1000), and lactyl-lysine (Cat. PTM-1401RM, PTM BIO, Chicago, IL, USA), and the mouse monoclonal antibodies against pan-CEA (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. Sc-48364, diluted 1:100), and CEACAM6 (Santa Cruz Biotechnology, Cat. sc59899, diluted 1:100) were used for Western blot analysis. Lysate preparation and immunoprecipitation were performed according to previously established methods [31 (link)]. In each reaction, 1 μg of the mouse monoclonal antibody against CEACAM6 (Santa Cruz Biotechnology, Cat. sc59899) was employed. After thorough washing, the immunoprecipitates were utilized for subsequent western blot analysis.

Protein lysates were separated based on molecular weight by SDS PAGE (Sodium dodecyl sulfate Pol-acrylamide Gel Electrophoresis), and electro blotted to 0.22 μm Nitrocellulose membrane (NCM) (Pall, USA). The transferred NCM was blocked in Tris-buffered saline containing 3% Bovine Serum Albumin (Sigma Aldrich, USA) and 0.2% Tween-20 (Sigma Aldrich, USA) and incubated in primary antibodies for pan AKT, phosphor-AKTs473, phosphor-AKTt308, phospho-c-Raf, phospho-GSK-3β, PTEN, phospho-PTEN, phospho-PDK1 (Cell Signaling Technology, USA) for overnight at 4 °C and then washed with TBST buffer. The blots were then incubated in Horse-radish peroxidase conjugated secondary antibody raised against rabbit or mouse (Sigma Aldrich, USA) diluted at 1:10,000 in blocking buffer for 1 hour in room temperature. The blots were then washed with TBST followed with TBS and the protein presence was probed through enhanced chemiluminescence, using Westar Supernova (Cynagen, Italy) and documented in ChemiDoc + XRS (Biorad, USA). The developed bands were analyzed using Image lab 5.1(Biorad, USA).