Anti β actin monoclonal antibody

Ang II, N-acetyl-L-cysteine (ROS scavenger), irbesartan (angiotensin receptor blocker), STO-609 (CaMKK inhibitor), compound C (AMPK inhibitor), KN-93 (CaMKII inhibitor), fluo-3 AM, 2′,7′-dichlorofluorescein diacetate (DCFDA), L-ascorbic acid, Claycomb medium, norepinephrine, gelatine, fibronectin, and monoclonal anti-β-actin antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). Monoclonal antibodies against ACC, CaMKII and CaMKIIδ and polyclonal antibodies against AT1R, TGF-β1, phosphorylated ACC, and phosphorylated CaMKII were purchased from Abcam (Cambridge, MA, USA). Monoclonal antibodies against NF-κB, phosphorylated JNK and AMPKα, and polyclonal antibodies against JNK and AMPKα were obtained from Cell Signaling Technology (Danvers, MA, USA). Foetal bovine serum (FBS), L-glutamine, and penicillin-streptomycin were acquired from Thermo Fisher Scientific (Foster City, CA, USA).

Immunoblot assay was performed, as previously described [25 (link)]. (1) anti-VEGF-A antibody (Abcam, Cambridge, UK), (2) anti-VEGFR2 antibody (Cell Signaling), (3) anti-type IV collagen antibody (Abcam), (4) monoclonal anti-human VASH2 antibody (clone 1760; provided by Tohoku University, Sendai, Japan), and (5) monoclonal anti-β-actin antibody (Sigma-Aldrich) were used as primary antibodies. Among these, anti-type IV collagen antibody was used under non-reducing condition. HRP-conjugated anti-rabbit or mouse IgG antibodies (Cell Signaling) were served as secondary antibodies. Images were obtained with ImageQuant LAS 4000 (GE Healthcare, Pittsburgh PA).

After treatment, T lymphocytes were harvested, washed twice in PBS and lysed with ice-cold RIPA buffer containing protease inhibitor on ice for 15 min. The lysates were centrifuged at 15,000 g for 15 min at 4 °C. The supernatants were collected and quantified for proteins concentration. Twenty-five µg proteins were separated on NuPAGE™ 4–12% Bis-Tris gels (Life Technologies, Carlsbad, CA). The separated proteins were transferred onto nitrocellulose membranes (GE Healthcare, Pittsburg, PA) and blotted according to standard procedures. Blots were probed with phosphorylated or total-ERK1/2 antibodies (Cell Signaling Technology). Monoclonal anti-β-actin antibody (Sigma-Aldrich) was used to visualize protein gel loading. Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Protein-antibody complexes were detected by enhanced chemiluminescence method using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) with a Chemi-Smart 5000 imager system (Vilber-Lourmat, Marne-la-Vallée, France). Blots were quantified by densitometry using ImageJ software.

cis-5,8,11,14,17-Eicosapentaenoic acid (EPA), metformin, compound C (AMPK inhibitor), insulin, STO-609 (CaMKK inhibitor), SB203580 (p38 MAPK inhibitor), fluo-3, AM and a monoclonal anti-β-actin antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). BAPTA-AM (intracellular calcium chelator), monoclonal antibody against ACC and polyclonal antibodies against GLUT4 were purchased from Abcam (Cambridge, MA, USA). Monoclonal antibodies against phosphorylated AMPKα, phosphorylated p38 MAPK, p38 MAPK, phosphorylated AS160, AS160, and the polyclonal antibody against AMPKα were obtained from Cell Signaling Technology (Danvers, MA, USA). The polyclonal antibody against phosphorylated ACC was provided by Merck (Rahway, NJ, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were acquired from Thermo Fisher Scientific (Foster City, CA, USA). The monoclonal antibody against c-Myc was acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

The antibodies and Chemicals used in this study included Hippo Signaling Antibody Sampler Kit (CST, #8579); GAPDH (CST, #5174); Anti-mouse IgG, HRP-linked Antibody (CST, #7076); Anti-rabbit IgG, HRP-linked Antibody (CST, #7074); YAP (D8H1X) XP® Rabbit mAb (CST, #14074); β-Catenin (D10A8) XP® Rabbit mAb (CST, #8480); Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (CST, #8889); Monoclonal Anti-β-Actin antibody (Sigma, A5316); Azacitidine (5-Azacytidine, AZA, 5Aza, S1782) and Verteporfin(S1786) were purchased from Selleck Biochem.

Cultured cells were collected in lysis buffer (5 mmol·L^-1 Tris, 5 mmol·L^-1 EDTA/EGTA, and proteinase inhibitors) and then ruptured by pipetting through a 20 gauge needle. Cell lysates were then centrifuged at 45 000 × g for 45 min. The pellet was resuspended in lysis buffer and the membrane protein was dissolved by addition of sodium dodecyl sulfate (SDS) to a 1% final concentration. Protein concentrations of SDS-dissolved lysates were determined by Micro BCA Protein Kit (Thermo Scientific, Rockford, IL, USA) and the lysates were used for Western blotting analysis. Each protein sample was boiled in SDS loading buffer, subjected to electrophoresis on a 10% SDS-polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. Membranes were incubated with a 1:300 dilution of affinity-purified anti-Cx43 antibody,^{73 (link)} or a 1:5 000 dilution of monoclonal anti-β-actin antibody (Sigma). Primary antibodies were detected with goat anti-rabbit IgG conjugated IRDye^® 800CW and goat anti-mouse IgG conjugated IRDye^® 680RD (1:15 000 dilution) using a Licor Odyssey Infrared Imager (Lincoln, NE, USA), as previously described.^{74 (link)} The band intensity was quantified by densitometry using Image J software (NIH, Bethesda, MD, USA).