Anti rabbit igg hrp

Twenty-microgram proteins were separated using 4–20% CRITERION® TGX Stain-Free™ Gels (300 V, 20 min) and subsequently transferred to Trans-Blot® Turbo™ Nitrocellulose membranes (both Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h at RT with 5% BSA in TBST (0.1% Tween20), before the primary antibodies were applied diluted 1:1000 in the same buffer (TrkA, Cell Signaling [2510]; pTrkA (Y674/675), Cell Signaling [4621S]; LMNA, Abcam [ab108595]; pLMNA (S22), Abcam [ab138450]; STMN1, abcam [ab52630]; pSTMN1 (S16), Abcam [ab47328]; pSTMN1 (S25), Abcam [ab194752]), except anti-Gapdh (EMD Millipore [MAB374]), which was diluted 1:2000. The membranes were incubated over night at 4 °C, washed with TBST and consecutively incubated with the appropriate secondary antibodies (anti-Rabbit IgG HRP, Thermo Scientific [31460]; anti-Mouse IgG HRP, Thermo Scientific [31430]) for 1 h at RT. Bands were visualized with SuperSignalTM Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and documented using a CHEMI SMART 5000 System (Vilber Lourmat, Eberhardzell, Germany).

Embryos were injected with 400 pg of Smad2 mRNA or Eomesodermin mRNA. A total of 100 embryos (injected or uninjected) were then processed as if for ChIP with either anti-Smad2 antibody (0.25 μg), anti-Eomesa antibody (1 ul) or a no antibody control. The immnuoprecipitated proteins were then eluted from the beads and subjected to SDS/PAGE. Western blotting was then performed with anti-Smad2 antibody (1:2,000) or anti-Eomesa antibody (1:600) then protein A-HRP (1:20,000; Merck Millipore, Billerica, MA, USA) or anti-Rabbit IgG-HRP (1:20,000; Thermo Fisher Scientific, Waltham, MA, USA).

Cell lysates were prepared using M-PER (Thermo Fisher Scientific, Waltham, MA, USA) buffer with protease inhibitor cocktail set III (Merck, Darmstadt, Germany). The contents of proteins in the lysates were determined using Bradford reaction. Samples (40 μg) were solubilized in Laemmli buffer added with 2% mercaptoethanol (Bio-Rad, Hercules, CA, USA) and were then subjected to 10% SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis as described earlier [21 (link)]. Following transfer, membranes were blocked for 1 h at room temperature in the presence of casein in TBS (tris-buffered saline)–1% Tween buffer (Bio-Rad) and subsequently incubated overnight at 4 °C with the following primary antibodies: anti-COX-2, anti-Nrf2, anti-FABP₄, anti-β-actin (GeneTex Inc., Irvine, CA, USA), and anti-PPARγ (Cayman Chemical, Ann Arbor, MI, USA), all of which were diluted to the ratio of 1:1000. After incubation, the membranes were washed and incubated with secondary antibodies (anti-rabbit IgG (HRP); Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Then, the membranes were washed again, and proteins were detected using a Clarity Western ECL Luminol Substrate detection kit (Bio-Rad). The integrated optical density of the protein bands was quantified using a Chemi Doc Camera with Image Lab software (Bio-Rad).