Horseradish peroxidase conjugated anti rabbit secondary antibody

OS cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing 1× proteinase inhibitor cocktail (Thermo Fisher Scientific) while on ice for 30 min. The lysates were then centrifuged at 12000 rpm for 10 min in order to collect the supernatant as total proteins. Protein concentrations were determined using the Bradford assay. Equal amounts of protein lysates (40 μg each) were separated using 4–20% SDS/PAGE and electro-transferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with TBST containing 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against PPP2R2A and β-actin. After washing, the membranes were incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibodies (Santa Cruz, U.S.A.) at room temperature for 2 h. After washing again, the target protein was detected via chemiluminescence using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

Glucose was obtained from Sigma (St. Louis, MO). ISL (PubChem CID: 638278) was sourced from Aladdin (Shanghai, China). The compound ISL dissolved in dimethyl sulfoxide and 0.5% sodium carboxyl methyl cellulose (CMC-Na) during in vitro and in vivo experiments, respectively. Antibodies against (TGF-β, sc-130348), Bcl-2-like protein 4 (Bax, sc-7480), B-cell lymphoma 2 (Bcl-2, sc-7382), extracellular signal-regulated kinase (ERK, sc-514302), phosphorylated ERK (p-ERK, sc-7383), Nrf-2 (sc-365949) and heme oxygenase-1 (HO-1, sc-136960) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against c-Jun N-terminal kinases (JNK, 9252 S), phosphorylated JNK (p-JNK, 4668 S), P38 (9212 S), phosphorylated P38 (p-P38, 9211 S), Cl-C3 (cleaved caspase 3, 9664), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5174 S) were obtained from Cell Signaling (Danvers, MA, USA). Antibodies against collagen type 4 (Col-IV, ab6586), TNF-α (ab1793), macrophage marker F4/80 (ab6640), and 3-nitrotyrosine (3-NT, ab61392) were obtained from Abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (sc-2357). One-step TUNEL apoptosis assay kit was obtained from Beyotime (Beijing, China).

Tissue sections obtained from xenografts were incubated with rabbit polyclonal anti-Ki-67 antibody at 4 °C overnight. Normal rabbit serum was used as a negative control. The cells were then incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz, CA, USA) at 37 °C for 1 h. A diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA, USA) was used to develop the signals.

Total proteins were extracted from peripheral blood mononuclear cell (PBMC) lysates from 25 patients with active RA and 10 HC. The proteins were separated by 10–12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). Immunoblots were performed using primary antibodies (1:1000 dilution) overnight at 4 °C against LC3-II (Abcam, Cambridge, MA, USA), p62 (Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with horseradish peroxidase-conjugated antirabbit secondary antibody (1:5000) for 1 h at 37 °C (Santa Cruz Biotechnology). The luminescent signal was detected by using the Fujifilm LAS-3000 image detection system (Fujifilm, Tokyo, Japan), and image processing and data quantification were performed using Multi Gauge version 2.02 software (Fujifilm). The LC3-II/LC3-I ratio was calculated as LC3-II expression levels divided by LC3-I expression levels, and the p62 expression levels were normalized to GAPDH.