Sb203580

Chemicals and reagents were obtained as follows: bortezomib (Bz) and carfilzomib (Cfz) from Selleckchem (Houston, TX, USA); Necrostatin-1, bafilomycin A1, N-acetyl-L-cysteine (NAC), and H₂O₂ from Sigma-Aldrich (St Louis, MO, USA); cycloheximide (CHX), SP600125, PD98059, and Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) from Calbiochem (EDM Millipore Corp., Billerica, MA, USA); Auranofin (AF) from AdipoGen (Liestal, CH, Switzerland); MitoTracker-Red (MTR), propidium iodide, and 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H₂DCF-DA) from Molecular Probes (Eugene, OR, USA); z-VAD-fmk from R&D systems (Minneapolis, MN, USA); Glutathione reduced ethyl ester (GSH-OEt) from Chemodex (Gallen, CH, Switzerland); SB203580 from Tokyo Chemical Industry (TCI, Tokyo, Japan); CDDO-ME from Cayman (Michigan, USA). The following primary antibodies were used: TrxR1 (HPA001395) from Sigma-Aldrich; Ubiquitin (sc-8017) and β-actin (sc-47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); ATF4 (#11815), p-eIF2α (#9721), eIF2α (#9722), and CHOP/GADD153 (#2895) from Cell Signaling Technology (Danvers, MA, USA); CHAC1 (15207-1-AP) from Proteintech (Rosemont, IL, USA). The secondary antibodies (rabbit IgG HRP(G-21234) and mouse IgG HRP (G-21040)) were from Molecular Probes.

Protein expression was determined in cell lysates from each treatment group at the same treatment manner with previous experiments. A 30-µg protein aliquot underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis and separated proteins were transferred to polyvinylidene fluoride membranes. The blots were blocked in 5% bovine serum albumin and probed with primary antibodies: anti-iNOS (ab15323), anti-COX2 (CST#2282), anti-phospho-ERK (CST#4376), anti-ERK (CST#4695), anti-phospho-JNK (CST#4668), anti-JNK (CST#9252), anti-phospho-p38 (CST#4511), anti-p38 (CST#9212), and followed by incubation with a horse radish peroxidase-conjugated secondary antibody. Protein bands were detected using a chemiluminescent substrate and images acquired using ImageQuant LAS 4000 (GE Healthcare, Buckinghamshire, UK). Relative protein band intensity was normalized to β-actin or GAPDH expression using ImageJ. SB203580 (Tokyo Chemical Industry, Japan) is a specific p38-α inhibitor and was used as a positive control for p38 phosphorylation inhibition.