Gapdh mouse monoclonal antibody

Total proteins were extracted from ovarian cancer cells using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were quantified using a BCA Kit (Beyotime Institute of Biotechnology). Equal protein was separated on an SDS-PAGE gel by electrophoresis and transferred onto a PVDF membrane. Then, 5% BSA was used to block the blots at 4 °C overnight. Then, membranes were incubated with the following primary antibodies: TDP1 mouse monoclonal antibody diluted at 1:500 (Santa Cruz Biotechnology, CA, USA: No. sc-365674), γ-H2AX (phospho-histone H2AX at ser139), rabbit polyclonal antibody diluted at 1:500 (Affinity Bioscience, Changzhou, China: No. AF3187), β-actin mouse monoclonal antibody diluted at 1:5000 (ZSGB Bio, Beijing, China: No. TA-09), and GAPDH mouse monoclonal antibody diluted at 1:5000 (Proteintech, IL, USA: No. 60004–1–1g). After washing, the membranes were incubated with peroxidase conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at 37 °C. The ECL system (ThermoScientific, Rockford, IL, USA) was used to visualize the blots. To detect γ-H2AX, we added phosphatase inhibitors to RIPA before protein collection. All assays were replicated three times.

A549 and H2228 cells were plated in six-well plates at a confluency of 70%. 48 hours after adenovirus infection, whole-cell extracts were prepared by lysing cells with the addition of 500 µL of hot SDS-PAGE buffer (Beyotime, P0015B). Tumor tissues were homogenized by TGrinder (Tiangen, OSE-Y30), and lysed with RIPA buffer containing complete protease inhibitor cocktail (Roche, 11697498001). Target proteins were detected by western blot analysis with the following antibodies: GAPDH mouse monoclonal antibody (Proteintech, 60004-1-Ig,), Akt (pan) (40D4) mouse monoclonal antibody (Cell Signaling Technology, 2920), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling Technology, 4060), p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Cell Signaling Technology, 4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, 4370), mouse monoclonal Anti-MAP Kinase, activated (Diphosphorylated ERK-1&2) antibody (Sigma-Aldrich, M8159), Ras Antibody (Cell Signaling Technology, 3965), and Anti-RAS (G12S) Mouse Monoclonal Antibody (NewEast Biosciences, 26186).

Cells were lysed using RIPA (Beyotime Institute of Biotechnology) buffer on ice for half an hour and went centrifuged at 17,000×g for 45 min at 4°C. The protein concentrations were set by the BCA protein assay kit (Beyotime Institute of Biotechnology). All cell lysates with 40 μg proteins were subjected to SDS-PAGE gels and electrophoretically blotted onto PVDF membranes. Membranes were blocked by TTBS (Tween-Tris-buffered saline) containing 5% non-fat milk for 2 h at room temperature and then incubated with primary antibodies as follows: PKM2 Rabbit Polyclonal Antibody (1:1,000, Cat#: ab137852, Abcam, Cambridge, UK), GLUT1 Rabbit Monoclonal Antibody (Cat# AF1015, 1:1,000, Beyotime Institute of Biotechnology, Jiangsu, China), GLUT3 Rabbit Antibody (Cat# bs-1207R, Bioss, Wuhan, China), LDHA Rabbit Monoclonal Antibody (Cat# AF1660, 1:1,000, Beyotime Institute of Biotechnology, Jiangsu, China), and GAPDH Mouse Monoclonal Antibody (1:5,000, Cat# 60004-1-lg, proteintech, USA). After that, membranes containing protein bands were incubated with HRP-polymerization secondary antibodies for 2 h at room temperature. The blots were visualized with ECL chemiluminescent detection system. The integrated density value (IDV) was calculated with software FluorChem2.0.

The primary antibodies were purchased from Proteintech Group Inc. (IL, USA), including CD31 rabbit polyclonal antibody (#11265-1-AP), VEGF rabbit polyclonal antibody (#19003-1-AP), Akt rabbit polyclonal antibody (#10176-2-AP), Akt-phospho-S473 mouse monoclonal antibody (#66444-1-Ig), HIF-1α rabbit polyclonal antibody (#20960-1-AP), and GAPDH mouse monoclonal antibody (#60004-1-Ig). The primary antibody against canstatin (#DF3550, rabbit polyclonal antibody) was obtained from Affinity Biosciences Inc. (OH, USA). The primary antibody against survivin rabbit polyclonal antibody (#GB11177) was purchased from Servicebio Inc. (Wuhan, China). The HRP-linked goat anti-mouse IgG secondary antibody (#SA00001-1) was purchased from Proteintech Group Inc. (IL, USA). The HRP goat anti-rabbit IgG secondary antibody (#7074) was purchased from Cell Signaling Technology Co. (Danvers, MA, USA). The primers used in this study were synthesized by Takara Biotechnology Co., Ltd (Dalian, China).

Cells were grown to about 90% confluency in 6 well plates, washed twice with ice cold PBS and resuspended in modified radio-immunoprecipitation (RIPA) lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-desoxycholate, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM PMSF). Cell suspensions were passed through a 0.45 μm needle 10 times and incubated for 15 min on ice, followed by sonication. The lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4°C. The samples were resuspended in 5× SDS sample buffer and heated at 98°C for 5 to 10 min. For western blot analysis, proteins were resolved in 12 or 15% SDS polyacrylamide gels and transferred to NC membrane (GE) using the wet blot transfer over 3 h (I = 300 mA). The blots were probed with primary antibodies, then washed with TBST (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20) three times and incubated with horseradish peroxidase-conjugated secondary antibody (Thermo). The blots were washed again with TBST and signals were visualized using chemiluminescence (ECL) system (GE, Cat. NO. RPN2232). The following antibodies were used: GFP mouse monoclonal antibody (Proteintech, Cat. No. 66002-1-Ig), GAPDH mouse monoclonal antibody (Proteintech, Cat. No. 60004-1-Ig).