Peroxidase conjugated anti rabbit igg secondary antibody

Western blot analysis was utilized to evaluate the expression of IL-1β, IL-6 and ICAM-1 in mouse gingival tissues. Briefly, palatal gingival tissues were collected (2.0 mm × 2.5 mm) from mice in all the three groups 2 h after transfection. Total protein was then extracted from each tissue using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and soluble proteins were resolved via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore, Darmstadt, Germany). The blots were subsequently probed with a primary antibody for IL-6 (1:1 000; Novus Biologicals, Littleton, CO, USA), IL-1β (1:1 000; Santa Cruz Biotechnology, Dallas, TX, USA) or ICAM-1 (1:1 000; Proteintech, Chicago, IL, USA). Equal protein loading was confirmed by probing for β-actin(1:2 000; Santa Cruz Biotechnology). For chemiluminescence detection, we used a peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10 000; Santa Cruz Biotechnology). The band intensities were measured using a ChemiDoc image analysis system (Fuji Film, Tokyo, Japan).

Total cellular protein was extracted using RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP 40, 0.5% deoxycholate, 0.1% SDS) supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN). After homogenization with a pellet pestle, the protein cell extracts were centrifuged at 12,000× g for 45 min, and the protein concentration of the supernatant was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins in cell lysates (25 μg) were resolved in 15% SDS–PAGE and electroblotted onto nitrocellulose membranes (Bio-Rad). Nonspecific binding to the membranes was blocked by incubation with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (TTBS) for 1 h at room temperature. Next, membranes were incubated with anti-caspase-3 (1:1000; #9662; Cell Signaling Technology) or anti-ß-actin (1:1000; #3700; Cell Signaling Technology) antibodies in 5% bovine serum albumin in TTBS overnight at 4 °C, followed by the corresponding peroxidase-conjugated anti-rabbit IgG secondary antibody (1:3000; sc-2357; Santa Cruz Biotechnology, Paso Robles, CA, USA) in 5% bovine serum albumin in TTBS for 1 h at room temperature. Peroxidase activity was detected on HyBlot CL autoradiography film (Denville Scientific, Plainfield, NJ, USA). Band intensities were quantified by densitometry using ImageJ software.

IMR32 and MRC5 cells were seeded and incubated with or without AE (50 μM). The collected cells were solubilized on ice in RIPA lysis buffer (SIGMA, Milan, Italy) supplemented with complete protease inhibitor cocktail tablet (Promega, Milan, Italy). Followed by centrifugation at 14 000 rpm for 5 minutes at 4°C. The protein concentration was determined by BCA assay (Pierce, Thermo Fisher), using BSA as a standard according to the manufacturer's instructions. Equal amount of total proteins (40 μg) were subject to Western blot analyses. The membranes were blocked with 5% w/v nonfat dry milk in PBS with 0.1% Tween‐20 and blots were probed with specific antibodies (Ab) to SSTR2, SSTR5 and GAPDH (Sigma, Milan, Italy). Blots were visualized with a peroxidase‐conjugated anti‐rabbit IgG secondary antibody (Santa Cruz, CA) and developed with enhanced chemiluminescence reagents (Pierce Thermo Fisher, Milan, Italy). Pixels intensity of the visualized bands was determined by using Image J software (W. S. Rasband, NIH, Bethesda, MD [http://rsb.info.nih.gov/ij/]). All experiments were conducted in triplicate.