Horseradish peroxidase conjugated anti rabbit igg secondary antibody

Whole-cell lysates were prepared using cell lysis buffer (Invitrogen) supplemented with a protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific), and then 30 µg of proteins were separated by polyacrylamide gel electrophoresis. After electrotransfer to polyvinylidene difluoride membranes, non-specific binding sites were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature. Membranes were incubated with anti-homeobox A7 (HOXA7) and anti-GAPDH primary antibodies (Abcam, Cambridge, MA, USA) overnight at 4°C, washed with TBST and then incubated with an anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (Abcam) for 1 hour at room temperature. Immune complexes were visualized using enhanced chemiluminescence. GAPDH was used as an internal control. The gray value of protein bands was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Total protein was extracted from PC cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific). In addition, protein concentration was detected by using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins (30 μg per lane) were separated by 10% SDS‑PAGE electrophoresis and then transferred onto a polyvinylidene fluoride membrane (PVDF, Millipore, Billerica, MA, USA). After that, membranes were blocked with 5% non-fat milk in Tween 20/Tris-buffered saline (TBST) at room temperature for 1 h and then incubated with the primary antibodies overnight at 4°C: anti-USP34 (1:1000, Abcam), anti-PRR11 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:1000, Abcam), anti-p-p38 (1:1000, Abcam), anti-p38 (1:1000, Abcam) and anti-β-actin (1:1000, Abcam). Later on, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (1:5000, Abcam) at room temperature for 1 h. Chemiluminescence Substrate Reagent Kit (Thermo Fisher Scientific) was used to develop signal on a ChemiDoc XRS + system (Bio-Rad, Berkeley, CA, USA). β-Actin was used as an endogenous control.

Briefly, total protein samples of heart, aortic arch, and abdominal aorta were extracted with the Tissue Protein Rapid Extraction Kit (Dingguo, Beijing, PR China) and were determined with the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Equal amounts of lysate proteins (100 mg) were loaded onto SDS-polyacrylamide gels (10% separation gels) and transferred to polyvinylidenefluoride membranes (EMD Millipore, Billerica, MA, USA) electrophoretically. After blocking with 5% nonfat milk in Tris-buffered saline (TBS) for 1 hour at room temperature, the membrane was incubated with angiotensin II (ANG II), endothelin 1 (ET-1), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (1:1,000, rabbit antibodies; Abcam, Cambridge, UK) overnight at 4°C. After washing with TBS containing 0.05% Tween-20 (TBST) for three times, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Abcam) for 1 hour at room temperature. After washing with TBST three times again, the blotted proteins were imaged through the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincon, NE, USA). The gray intensity of proteins were counted using ImageJ software.