Goat anti rabbit igg hrp conjugated secondary antibody

ELISA was performed to quantify the cytokine levels. Mice (n = 5 per time point per group) were sacrificed by decapitation at 3 and 8 weeks post-induction and peripheral blood was collected at 4°C. Heparin was added to samples to prevent coagulation. The blood samples were first centrifuged at 1,000 × g for 20 min, then at 10,000 × g for 10 min at 4°C, to ensure complete platelet removal. Plasma samples were first incubated in 96-well plates precoated with interleukin 6 (IL-6), reactive oxygen species (ROS), and γ-aminobutyric acid (GABA; R&D Systems, Minneapolis, MN, USA) primary antibodies for 1 h at 37°C, and then were subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:2000; Biorad, CA, USA) for 1 h at 37°C. The concentration of bound proteins was measured using a microplate reader (model 680, Bio-Rad Laboratories, Corston, UK) at 450 nm and analysis was performed using the GraphPad Prism version 4 software (GraphPad Prism Software, Inc, CA, USA).

For detection of Chd4, Znf219, troponin I2, fast skeletal type (Tnni2), and troponin I3, fast skeletal type (Tnnt3), heart tissue was homogenized in lysis buffer (50 mM Tris pH 8, 0.42 M NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors (pepstatin, leupeptin, aprotinin, and PMSF) using a Magnalyzer apparatus. The resulting homogenate was incubated for 60 min at 4 °C on a rocking platform and then centrifuged at 14,000× g for 15 min, and the supernatant was recovered. Equal amounts of protein were resolved by 6% or 10% SDS-PAGE, and proteins were transferred to 0.22 µm nitrocellulose membranes. Blots were probed with polyclonal primary antibodies against Chd4 (1:1000 dilution; ab72418, Abcam, Cambridge, UK), Znf219 (1:1000 dilution; ab71279, Abcam), Tnni2 (1:1000 dilution; ab119943, Abcam, Cambridge, UK), or Tnnt3 (1:1000 dilution; sc-20643; Millipore, Bedford, USA), followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (Bio-Rad) and detection by enhanced chemiluminescence (Amersham, Los Angeles, CA, USA). As a loading control, membranes were re-probed with a primary antibody against tubulin (mouse monoclonal, 1:40,000 dilution; T6074, Sigma) or PSF (mouse monoclonal, 1:1000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) (Table 2).

The mouse anti-FLAG (M2) and anti-Actin monoclonal antibodies were obtained from Sigma-Aldrich (St Louis, MO). Mouse monoclonal anti-MYC and anti-Ras (detects endogenous Ras) antibodies were generated in house. Rabbit polyclonal anti-TβRI, anti-TβRII and mouse monoclonal anti-H-Ras antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-FLAG antibody was obtained from ABR (Affinity BioReagents, Golden, CO). Rabbit polyclonal anti-phospho-Smad2 antibody was kindly provided by Prof Peter ten Dijke (Leiden University Medical Center, Netherlands). Mouse monoclonal anti-Smad2 antibody was obtained from BD Transduction Laboratories (Rockville, MD). Goat anti-mouse IgG HRP conjugated secondary antibody, Goat anti-rabbit IgG HRP conjugated secondary antibody were obtained from Bio-Rad (Bio-Rad Laboratories, Gladesville, N.S.W., Australia). The anti-mouse Alexa⁴⁸⁸ and Alexa⁵⁴⁶-conjugated secondary antibodies were from Invitrogen (Invitrogen Corp., Mulgrave, Australia). Human recombinant TGF-β1 was obtained from R&D Systems (Minneapolis, MN). Doxycycline and Cycloheximide were purchased from Sigma-Aldrich, while MG132 was obtained from Merck (Merck, Darmstadt, Germany).