Ab8932

Apatinib mesylate (Heng Rui) were grinded into powder and dissolved 0.5%CMC (Solrbio). Pemetrexed (Qi Lu) were dissolved 0.9% saline. Primary antibodies against AKT (ab8805), phospho‐AKT (ab8932), ERK (ab54230), phospho‐ERK (ab201015), mTOR (ab2732), phospho‐mTOR (ab84400), MEK (ab178876), phospho‐MEK (ab194754), HIF‐1α (ab51608), CD31 (ab28364), α‐SMA (ab5694), collagen IV (ab6586), MMP2 (ab37150), MMP‐9 (ab38898), and β‐actin (ab8227) were purchased from abcam. Primary antibodies against cleaved‐caspase3 (9664), ki67 (9449), and anti‐rabbit or anti‐mouse IgG horseradish peroxidase (HRP)‐linked secondary antibodies were purchased from Cell Signaling Technology.

Apatinib mesylate standard products were provided by Jiangsu Heng Rui Pharmaceutical Co. Ltd. (Jiangsu, China) and icotinib standard product was obtained from Zhejiang Beida Pharmaceutical Co., Ltd. (Zhejiang, China), pemetrexed was provided by Qilu Pharmaceutical Co., Ltd. (Shandong, China) and osimertinib standard was purchased from Selleck.cn. Three cell lines A549, HCC827 and H1975 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI media (HyClone) containing 10% fetal bovine serum (FBS, Gibco),100 U/mL penicillin (HyClone) and 100 ug/mL streptomycin (HyClone). Primary antibodies against AKT(ab8805), phosphor‐AKT(ab8932), ERK(ab54230), phospho‐ERK(ab201015), CD31(ab28364), mTOR(ab2732), phosphor‐mTOR(ab109268) and GAPDH(ab181602) were purchased from abcam (Cambridge, MA, USA). Primary antibodies against ki67 (9449) and anti‐rabbit or anti‐mouse IgG horseradish peroxidase (HRP)‐linked secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Protein extracts from tumor tissue samples were prepared by suspending the cells in a cell lysis buffer (Sigma) and protease inhibitor cocktail (Roche). Each extract was prepared as above and an equivalent to 20 μg total protein was separated by SDS-PAGE.
Annexin II Antibody (ab41803, Abcam©, France); hENT1 (antibody LS-C178673, LSBio©, France); AKT (ab8932, Abcam©, France); ERK-p (9101S Cell signaling, France); Raf-1 (ab173539, Abcam©, France); mTOR (ab1093, Abcam©, France); Caspase-3 (ab47131, Abcam©, France). Horseradish peroxidase-conjugated anti-mouse (Promega W402B 28570702©, France) or anti-rabbit IgG (w401B 29303402© Promega, France) was used to detect specific proteins.
Detection of specific proteins was carried out using an enhanced chemiluminescence western blotting kit (Pierce).
We measured the intensity of each band using LAS 4000 software and calculated the relative protein levels normalized to that of the β-actin antibody (A5316-2ML SIGMA ©, France).

Antibodies to USP13 (ab99421), NF-kB p65 (ab16502), NF-kB phosph-p65 (ab86299), pan-AKT (ab8805), phosph-AKT (Ser473) (ab8932), phosph-AKT (Thr308) (ab8933), PTEN (ab170941) and GAPDH (ab181602) (all from abcam) were used according to the manufacturers’ protocols. The western blotting analysis was performed as described before. Briefly, equal amounts of protein extracts were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20) with 5% (w/v) non-fat dry milk and were then incubated with primary antibodies in TBS-T at 4 °C overnight. After three washes with TBS-T for 15 min each, the membranes were incubated with the appropriate HRP-labeled secondary antibodies for 1 h at 37 °C. The immunobands were visualized using the ECL reagents (Transgen Biotechnology, Beijing, China) on a MicroChemi Chemiluminescent Imaging System (DNR Bio-Imaging Systems, Mahale HaHamisha, Jerusalem, Israel). The densitometric values for each band were calculated by Image J 1.46r software (Wayne Rasband, National institutes of Health, Bethesda, MA, USA), and the statistical analysis were conducted based on the ratios of target protein/GAPDH.

qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-it^TM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2^-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.

After 48 hours of transfection, the cells in six-well plates were collected and placed on ice. To extract the proteins, RIPA lysate with protease inhibitor was used. BCA method was used to determine the protein concentration. Then we added about 20 μg protein to each well of a vertical electrophoresis tank after being heated at 95 °C for 5 min. Following that, the protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the current study were list as follows: ARHGEF39 (1:1000, cat.no. ab67211, Abcam), AKT (1:500, cat. no. ab64148, Abcam), p-AKT (1:500, cat.no. ab8932, Abcam), ERK (1:1000, cat.no. ab32537, Abcam), p-ERK (1:1000, cat.no. ab131438, Abcam). Following that, the membrane was rinsed with TBST 35 min and incubated with suitable secondary antibodies at ambient temperature. Then the protein bands were washed and developed with enhanced chemiluminescence western blot detection kit. The gray value was scanned by the QUANTITY ONE software and the relative expression of each protein was calculated with GAPDH as the internal reference.