Anti rat igg hrp

To perform dot blot, RNA was treated with TURBO DNase (ThermoFisher) following the manufacturer’s protocol. As a control, RNA was treated with 1U RNase A (Qiagen) for 1 h at 56 °C. RNA was then loaded onto a Hybond-N + membrane (GE Healthcare), allowed to air dry and crosslinked twice at 200,000 μJ/cm² UV. To control for RNA loading, the membrane was then incubated with 0.04% methylene blue in 0.5 M sodium acetate for 5 min, following rinsing with PBS 0.1% Tween-20, it was imaged using the colorimetric function of ChemiDoc Imaging System (BioRad). The membrane was then blocked in 3% w/v skim milk PBS-0.1% Tween-20 for 1 h at room temperature, incubated in blocking buffer with 1:500 rat anti-5 hmC (Diagenode) overnight at 4 °C and followed by incubation with 1:1000 anti-rat IgG HRP (ab6734, Abcam) in blocking buffer. 5 hmC detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) and imaged on a ChemiDoc Imaging System (BioRad).

Western blotting was performed as described previously (Mahmood and Yang, 2012 (link)). Briefly, cells or myocardial tissues were collected and homogenized, and total proteins were isolated using radioimmunoprecipitation assay buffer (Beyotime, China) according to the manufacturer’s protocol. Equal amounts of protein (20 µg/lane) from each sample were separated using SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked with 5% BSA solution for 2 h at 28°C, incubated with primary antibodies at 4°C overnight, and then hybridized with secondary antibodies at 28°C for 2 h. The membranes were stained using an ECL kit (Thermo) and analyzed using ImageJ software. GAPDH was used as an internal control. The following antibodies were used: primary antibodies against FTH1 (1:1,000, Abcam), GPX4 (1:1,000, Abcam), ACSL4 (1:1,000, Abcam), LPCAT3 (1:1,000, Abcam), PTGS2 (1:1,000, Abcam), NRF2 (phospho S40) (1:1,000, Abcam), HIF (1:1,000, Abcam), lamin B1 (1:5,000, Abcam), and GAPDH (1:5,000, Abcam) and secondary antibodies for anti-Rat IgG (HRP; 1:5,000, Abcam) and anti-mouse IgG (HRP; 1:5,000, Abcam).