Anti mouse igg hrp

The primary antibodies of TEAD1 (sc-376113), TEAD4 (sc-134071), and CTGF (L-20) (sc-14939) were from Santa Cruz (Dallas, TX, USA). YAP1 (ab52771) antibody and HA tag (ab18181) were achieved from Abcam (Cambridge, MA, USA). AMOTL1 (HPA001196) and Flag-tag (F3165) antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other primary antibodies were from Cell Signaling (Danvers, MA, USA), including p21 (#2946), p27 (#2552), pRb (Ser807/811) (#9308), Erk (#9102), pErk (#9101), Myc (#2278), cyclin D1 (#2978), c-Myc (#9402), and GAPDH (#2118). Anti-Mouse IgG-HRP (Dako, Glostrup, Denmark, 00049039, 1:30,000) and anti-Rabbit IgG-HRP (Dako, 00028856, 1:10,000) were used for secondary antibodies. The related protocol was suggested before [13 (link)].

Total protein was extracted from gastric cancer cell lines and paired primary tumors and non-tumorous tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA). Twenty-microgram of protein mixed with 2 × SDS loading buffer were loaded on each lane, separated by 12% SDS-polyacrylamide gel electrophoresis. YY1 protein was then detected using anti-YY1 antibody (1:1000, H-10, sc-7341, Santa Cruz Biotechnology, Dallas, TX). Other antibodies applied included cleaved-PARP (Asp214) (1:1000, #9541, Cell Signaling, Danvers, MA), active-β-catenin (1:1000, #05-665, Millipore, Billerica, MA), β-catenin (1:10000, #610154, BD Transduction Laboratories, San Jose, CA), CCND1 (1:1000, #2926, Cell Signaling, Danvers, MA ), c-Myc (1:1000, #9402, Cell Signaling, Danvers, MA), anti-Mouse IgG-HRP (1:30000, #00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:40000, #00028856, Dako, Glostrup, Denmark).

An equal amount of protein (20 µg as determined by Bradford Protein Assay) for each sample was separated by gel electrophoresis using 10% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and Mini Trans-Blot Cell (Bio-Rad). The proteins were blotted onto nitrocellulose membranes (Bio-Rad; catalog#1620146) by Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were then incubated overnight at 4°C with the primary antibodies as follows (catalog#): anti-p-p65 (3033), anti-p-c-Jun (9261), anti-p-p38 (9211) and p-ERK1/2 (9101) purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies were detected by 1-hour incubation at room temperature using the secondary antibody anti-rabbit immunoglobulin G (IgG)-HRP (catalog#7074; Cell Signaling Technology). Protein bands were visualized using Clarity Western ECL Substrate (Bio-Rad) and C-DiGit Blot Scanner (LI-COR, Lincoln, NE, USA). Following which, the membranes were stripped and reprobed with β-actin (catalog# A-1978; Sigma-Aldrich) and anti-mouse IgG-HRP (catalog# p0447; Dako, Glostrup, Denmark). Densitometric analysis of the band and background intensities were measured by Image Studio Digits Version 3.1. The results were normalized to the β-actin levels and shown as fold changes relative to the unstimulated and untreated samples (vehicle).

Protein was extracted in ice-cold RIPA lysis buffer containing complete protease inhibitor cocktail tablets (Roche, Rotkreuz, Switzerland). RASAL2 was detected with a polyclonal anti- RASAL2 antibody (1:250, ab121578, Abcam). Anti-YAP1 antibody (1:20000, ab52771) was provided by Abcam. Other primary antibodies were from Cell Signaling (Danvers, MA, USA), including antibodies to LATS2 (1:1000, #5888S), phospho-YAP1 (S127) (1:1000, #4911) and Cyclin D1 (1:1000, #2926). β-actin (1:100000, A5441, Sigma-Aldrich, St. Louis, MO, USA) expression was used as an equal loading control. The secondary antibodies were anti-Mouse IgG-HRP (1:15000, 00049039, Dako, Agilent Technologies, Santa Clara, CA, USA) and anti-Rabbit IgG-HRP (1:5000, 00028856, Dako).

Protein was extracted from gastric cancer cell lines and paired primary tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA) and 20 μg of protein mixed with 2 × SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis. Protein expression was detected using primary monoclonal anti-AKT2 antibody (1:1000 dilution, #3063, Cell Signaling, Danvers, MA), anti-phospho-AKT (S473) (1:1000 dilution, #9271, Cell Signaling) and anti-pS6 antibody (1:1000, #4858, Cell Signaling). The secondary antibodies were anti-Mouse IgG-HRP (1:30000, 00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:10000, 00028856, Dako). The Western blot bands were quantified by ImageJ.

Western blot was performed with whole-cell protein lysates in RIPA lysis buffer. Equal amounts of 10 µg proteins were used for western blot analyses. PHLDB2 primary antibody was from Abcam (1:1000, ab202350, Cambridge, UK). Other primary antibodies were from Cell Signaling (Danvers, MA, US), including NOTCH3 (D11B8) (1:1000, #5276), p21 (1:1000, #2946), p27 (1:1000, #2552), Phospho-Rb (Ser807/811) (1:1000, #9308), cleaved caspase-3 (Asp175) (1:1000, #9661), cleaved-PARP (Asp214) (1:1000, #9541), Phospho-Akt (Ser473) (1:1000, #4060), Phospho-mTOR (Ser2448) (1:1000, #5536), CCND1 (1:1000, #2978), CDK4 (1:1000, #12790), CDK6 (1:1000, #3136), and GAPDH (1:2000, #2118). Secondary antibodies anti-Mouse IgG-HRP (1:30000, 00049039) and anti-Rabbit IgG-HRP (1:10000, 00028856) were from Dako (Glostrup, Denmark).