Rabbit anti caspase 3

Subcellular fractionations were carried out as previously described with modifications.⁴⁰ The purity of each fraction was further validated by using anti‐rabbit TLR‐4 (Santa Cruz, sc‐10741; membrane marker), anti‐rabbit Caspase‐3 (Cell Signaling, 9662S; cytosolic marker), and anti‐rabbit Histone H3 (Cell Signaling, 4499s; nuclear marker).⁴¹ Western blot was performed on flash snap‐frozen human myocardium tissues as we previously published.⁴⁰, ⁴¹ The below primary antibodies were used: anti‐rabbit TFR‐1 (Cell Signaling, 13208s); anti‐rabbit FPN (Novus, NBP1‐21502); anti‐rabbit FTN (Abcam, ab75973); anti‐mouse DMT‐1 (Abcam, ab55735), followed by incubation with HRP‐conjugated secondary antibodies at 1/5000 dilution (Cell Signaling). The total protein loadings were visualized by MemCode reversible stain (24585, Thermo Scientific) as a loading control. Fiji ImageJ software (NIH, Bethesda, MD) was used for band intensity quantitation.

p62, LC3, caspase-3, and SLC7A11 protein levels were analyzed by western blotting. The cells were treated with 2.5 mM Hcy or 2.5 μM chloroquine (CQ) for 24 h, washed with Dulbecco's phosphate buffered saline (DPBS), and lysed in lysis buffer [50 mM HEPES (pH 7.4), 5 mM EDTA, 120 mM NaCl, 1% Triton X-100, protease inhibitors (10 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin) and phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate)]. The lysate was centrifuged at 10,000×g for 15 min and 15 μg of protein in the supernatant was resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the following primary antibodies: anti-rabbit LC3 polyclonal antibody (Novus Biologicals, Centennial, CO, USA), anti-rabbit p62 (Cell Signaling Technology, Danvers, MA, USA), anti-rabbit caspase-3 (Cell Signaling Technology), anti-rabbit SLC7A11 (Cell Signaling Technology), and anti-mouse β-actin polyclonal antibody (Sigma-Aldrich). Following primary antibody incubation, the membrane was incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies. Chemiluminescence was detected with Immobilon (Merck, Darmstadt, Germany).