Rabbit igg antibody

K36 was synthesized by the author, Professor Yueh-Hsiung Kuo [21 (link)], and was dissolved in dimethyl sulfoxide (DMSO) for experimentation. The final concentration of DMSO was lower than 0.05% and did not cause cytotoxicity. The Bradford reagent was purchased from Bio-Rad Laboratories (Hercules, CA, USA). DCFDA, DMSO, leupeptin, and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sodium dodecyl sulfate (SDS), IGEPAL CA-630, MTT, Tris, and Tween20 were obtained from USB Corporation (Cleveland, OH, USA). Fetal bovine serum (FBS), penicillin–streptomycin, trypsin–EDTA, and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). TIMP-1 (GTX112096), IL-6 (GTX22105), HO-1 (GTX101147), and rabbit IgG antibody were purchased from Genetex (Beverly, MA, USA). ERK-1 (sc-93), MMP-1 (sc-12348), MMP-2 (sc-13595), Bcl-2 (sc-7382), c-Fos (sc-7202), COX-2 (sc-19999), JNK (sc-46006), and β-Actin (sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A western blot detection reagent and PageRuler prestained protein ladder were purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, England). All other chemicals used in this study were of reagent grade.

Autophagy flux was analyzed by confocal microscopy (Nikon C2 Si+) using DQ™-Green BSA (self-quenched green BODIPY dye conjugated to BSA; Molecular Probes). H9c2 cells were placed on 12-mm coverslips; exposed under IH for 72 h; incubated with DQ™-Green BSA (10 μg/ml) in DMEM under IH at 37°C for additional 24 h; washed twice with N-2-hydroxy-ethylpiperazine-N′-2-ethanesulphonic acid (HEPES)-buffered Tyrode solution (NT) composed of 10 mM HEPES, 11 mM glucose, 140 mM NaCl, 4.5 mM KCl, 2.0 mM CaCl₂, 1.2 mM KH₂PO₄, and 1.0 mM MgCl₂ with pH adjusted to 7.4 with NaOH at 37°C buffer to remove excess probe; fixed with 10% formaldehyde at RT for 1 h; and then permeabilized with 0.15% Triton X-100 at RT for 15 min. To block the nonspecific binding sites, cells were treated with 5% dried non-fat milk in NT buffer at RT for 15 min. After blocking, cells were incubated with anti-LC3 antibody (Cell Signaling, MA, USA) at a 1:200 dilution overnight at 4°C, washed twice with NT buffer, and then incubated with rabbit IgG antibody (Dylight 594, Gene Tex, San Antonio, TX, USA) at a 1:200 dilution.