Peroxidase conjugated anti mouse igg

MEPM cells were plated onto a 60-mm dish at a density of 50,000 cells per dish and treated with either 30 μM atRA or vehicle for 72 h, or with each miR mimic or control miR for 48 h. The treated cells were lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, United States) with a protease inhibitor cocktail (Roche, Indianapolis, IN, United States). The cells were harvested and centrifuged at 21,130 × g for 10 min at 4°C. The supernatant of each sample was collected, and protein concentration was determined using the BCA protein kit (Pierce). Protein samples were applied to Mini-PROTEAN TGX Gels (Bio-Rad, Hercules, CA, United States) and transferred to a polyvinylidene difluoride (PVDF) membrane. A mouse monoclonal antibody against GAPDH (MAB374, Millipore, Burlington, MA, United States, 1:6,000), a rabbit monoclonal antibody against CCND1 (2978, Cell Signaling Technology, 1:1,000), and a rabbit polyclonal antibody against cleaved caspase 3 (9661, Cell Signaling Technology, 1:1,000) were used. Peroxidase-conjugated anti-mouse IgG (7076, Cell Signaling Technology, 1:100,000) and anti-rabbit IgG (7074, Cell Signaling Technology, 1:100,000) were used as secondary antibodies.

Acinar cells were isolated and seeded into 6-well tissue culture plates. The cells were treated with 250 µM hydrogen peroxide for 7.5 min with or without 10 and 30 µM TCT pretreatment (1 h). Total protein lysates were separated on 8% SDS-PAGE gels at 100 V for 90 min. Proteins were transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in phosphate buffered saline with Tween^® 20 (PBST) for 1 h at room temperature. Membranes were incubated with antibodies against PAR (clone 10H; purified in-house) overnight at 4 °C. After washing with Tris buffered saline with Tween^® 20 (TBST), membranes were incubated with peroxidase-conjugated anti-mouse IgG (1:3000; Cell Signaling, Danvers, MA, USA) and anti-β-actin (1:20,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies for 2 h at room temperature. Membranes were then washed with PBST and incubated with WestFemto chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA, USA). Luminescence was detected with a Chemidoc Touch gel documentation system (BioRad Laboratories, Hercules, CA, USA). Signal intensities were quantified using Image Lab software (BioRad). The blot area used for signal quantification was selected to cover the PARylated PARP1 region (ca. 100–180 kDa) and actin signals were used for normalization.

Western blot was performed as described previously [30 (link)]. Antibody concentrations of primary antibodies were used as follows: Anti-DR4 (1:1000; Pro Sci, Fort Collins, CO, USA), anti-caspase-8 (1:1000; Enzo), anti-CDK9, anti-PARP, anti-FADD, anti-Bax, anti-Bak, anti-Mcl-1, anti-Bcl-xL, anti-Bcl-2, anti-cFLIP (AdipoGen, San Diego, CA, USA), anti-cIAP1, anti-cIAP2, anti-XIAP, anti-survivin (1:1000; Cell Signaling Technology), anti-caspase-9 (1:500; Cell Signaling Technology), anti-DR5, anti-Bid (1:2000; Cell Signaling Technology), anti-β-actin (1:5000; Sigma-Aldrich), anti-caspase-3 (1:2000; R&D, Minneapolis, MN, USA), anti-RNA polymerase II total (RNA Pol II) (1:2000), anti-pSer2 RNA Pol II (1:5000; Covance, Princeton, NJ, USA). Immuno-complexes were detected using peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG (1:5000; Cell Signaling Technology) followed by chemiluminescence detection (Pierce ECL Western Blotting Solution; Thermo Fisher Scientific).

HUVECs were exposed to the EP4 antagonist (AH23848; 100 μM, Sigma, Kanagawa Prefecture, Japan) with or without L-902,688 (1 µM) or TGF-β (5 ng/mL) for 24 h. Western blotting was performed using anti-eNOS (BD), anti-E-cadherin (E-cad) (Cell Signaling, Danvers, MA, USA), anti-CD146 (Abcam, Cambridge, UK), anti-α-SMA (Thermo, Waltham, MA, USA), and anti-Twist (Novus, Frauenfeld, Switzerkand) primary antibodies. Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling) were administered as secondary antibodies. Blots were visualized using the enhanced chemiluminescence detection system (Amersham, UK). Samples were normalized to GAPDH (Santa Cruz, CA, USA) and quantified by densitometry.

The isolation of cytosolic protein was performed by scraping cells in lysis buffer (25 mm Tris–HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, 0.1 mm NaF, and 0.1 mm Na₃VO₄). The protein concentration was determined by the Bradford method using the Bio-Rad reagent (Bio-Rad Laboratories, Hercules, CA, USA). 20 μg of protein extract were subjected to 10% SDS-PAGE and electroblotted on PVDF membranes, which were blocked at room temperature for 1 h in blocking solution (5% BSA in TBS-T), and incubated overnight at 4 °C with anti-ubiquitin (1 : 1000; Abcam). The membrane was incubated with peroxidase-conjugated anti-mouse antibodies (1 : 10 000; Amersham Biosciences). Proteins were revealed by using an ECL kit (Millipore) after 10 s exposure. Normalization was performed with anti-tubulin (1 : 1000 in TBS-T; Cell Signaling) for 1 h at room temperature and incubated with peroxidase-conjugated anti-mouse IgG (1 : 10 000 in TBS-T). Proteins were revealed as above. Band intensity was quantified by using Image Lab Software ChemiDoc (BioRad).