Anti cdk2

Rabbit anti-NCKAP1 was purchased from Proteintech. Rabbit anti-WASF1, anti-pRb, anti-P53, anti-cyclin D1, anti-p27, anti-CDK2, and anti-CDK4 antibodies and mouse anti-p18 and anti-CDK6 antibodies were purchased from Cell Signaling Technology.

Western blot was employed to determine the regulated gene by Rab14 in the AKT pathway. Total proteins were extracted with RIPA lysis buffer (Wolsen, China) from cells harvested 48 hours after transfection, and were separated by 10% SDS polyacrylamide gels. They were then electrophoretically transferred to polyvinylidene difluoride membrane (Millipore, USA). After the samples were incubated with primary antibodies at 4°C overnight and secondary antibodies for 2h at room temperature, then were visualized with enhanced chemiluminescence detection system (UVP, USA). The primary antibodies for the differentially expressed gene used were as following dilutions: rabbit polyclonal anti–Rab14 (working dilutions: 1:200, #15662-1-AP, Proteintech, China), rabbit mAb anti-Akt (working dilutions: 1:1,000, #9272, Cell Signal Technology, USA), rabbit mAb anti–phospho-AktSer473 (; working dilutions: 1:2,000, #4060, Cell Signal Technology, USA), rabbit mAb anti-CCND1 / p-CCND1 (working dilutions: 1:1,000, #2978 / #3300, Cell Signal Technology, USA), rabbit mAb anti-CDK2 (working dilutions: 1:500, #2546, Cell Signal Technology, USA), rabbit mAb anti-Bax (working dilutions: 1:1,000, #5023, Cell Signal Technology, USA) and mouse monoclonal anti-GAPDH (working dilutions: 1:2,000, #8795, Sigma-Aldrich, USA).

Western blot analysis was carried out as previously interpreted [23 (link), 24 (link)]. Primary antibodies in this experiment comprised of: anti-Sirt6 (Sigma), anti-phosphor-PI3 Kinase p110α, anti-phospho-AKT(Ser473), anti-total pan-AKT, anti phospho-mTOR, and anti-total pan-mTOR, anti-PTEN, anti-phospho-4EBP1, anti-FoxO1, anti-HIF-1α, anti-PARP [specific to the full-length (116 kDa) and the cleaved form (89 kDa) of PARP], anti-p27, anti-CDK2, anti-phospho-ATM, anti-phospho-ATR, anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68) and anti-β-tubulin (Cell Signaling Technology, MA, USA), β-actin (Zhongshan Goldenbridge, Beijing, China). Details are shown in Supplementary methods.

Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein concentration was quantified using a BCA Protein Assay kit (Pierce, USA). Total protein was separated using 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, USA). Western blot analysis followed a standard procedure. After incubated with the primary antibodies anti-WTAP, anti-CDK2 or anti-GAPDH (Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.

Total cell lysates were prepared in 2× sample loading buffer (250 mM Tris-HCl (pH 6.8), 10% glycerol, 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 0.006% bromophenol blue, 5 mM sodium orthovanadate, and 50 mM sodium fluoride; Bio-Rad, Hercules, CA, USA). Protein concentrations were quantified through the BCA method [24 (link)] using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (5–20 µg) were separated using 6–13% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA (Sigma-Aldrich) and probed with anti-phospho-STAT3 (Y705), anti-STAT3, anti-cyclin E, anti-CDK2, anti-cyclin D1, anti-CDK4, anti-CDK6, anti-survivin, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP (D214), and anti-vimentin antibodies (Cell Signaling Technology, Beverly, MA, USA); anti-β-actin horseradish peroxidase antibody (Santa Cruz Biotechnology, Dallas, TX, USA); anti-E-cadherin and anti-N-cadherin antibodies (BD Biosciences, San Jose, CA, USA); or anti-Ki-67 antibody (Abcam, Cambridge, UK). The blots were detected using the WEST-Queen detection system (iNtRON Biotechnology, Seongnam, Korea) [25 (link)].

TSL was purchased from Pufei De Biotech (Chengdu, China). Endothelial cell medium (ECM) was purchased from Sciencell (USA). Fetal bovine serum (FBS) was purchased from PAN Biotech (Germany). Recombinant human vascular endothelial growth factor (VEGF165) was obtained from PeproTech (USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). EdU kit was purchased from Ribobio (GuangZhou, China). Anti-phospho-VEGFR2, anti-VEGFR2, anti-phospho-JNK, anti-JNK antibody, anti-phospho-ERK1/2, anti-ERK1/2 antibody anti-phospho-p38, anti-p38 antibody, anti-CDK2, anti-CDK4, and anti-tubulin were purchased from Cell Signaling Technology (USA). Anti-MMP2 and anti-MMP9 were purchased from Abcam (UK).

b-AP15 was obtained from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO at a stock concentration of 10 mM, aliquoted and stored at −80°C. Other agents are N-acetyl-L-cysteine (NAC, Sigma-AldrichInc., St. Louis, MO); pan caspase Inhibitor Z-VAD-FMK (EnzoLife Sciences International, Inc, Plymouth Meeting, PA). Antibodies (Abs) used in this study were purchased from following sources: anti-CDK2, CDK4, CDK6, anti-cyclinD1, anti-p27, anti-PARP, anti-Bcl-2 (50E3), anti-Bim (Y36), anti-Noxa (D8L7U), anti-BIP (C50B12), anti-CHOP (L63F7), eIF2α, phospho-eIF2α (Ser51), anti-HSP70, anti-HSP90, anti-AIF, cytochrome C and anti-Phospho-MDM2 (Cell Signaling Technology, Beverly, MA, USA); anti-Rb, phospho-Rb (S780), anti-cleaved caspase-8 (Cleaved Asp384) (Assay biotechnology Company, Inc); anti-cleaved caspase-9 p35 (D315), anti-cleaved caspase-3 (p17), anti-AR, anti-MDM2 and anti-GAPDH (Bioworld Technology, Inc). anti-p53(Abcam), anti-ubiquitin (P4D1), anti-K48-linked tetra-ubiquitin, Bax (B-9) (Santa Cruz Biotechnology, Santa Cruz, CA); MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). CCK-8 assay kit, PI and Annexin V-FITC apoptosis Detection Kit, DCFH-DA and cell apoptosis Rhodamine 123 Detection Kit were purchased from Keygen Company (Nanjing, China). Dynabeads antibody coupling kit was from Life technologies.