Bca protein assay kit

Sodium hydrosulfide (NaHS; a donor of H₂S) was purchased from Gibco-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS), 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), 740 Y-P (a PI3K agonist), LY294002 (a reversible PI3K inhibitor), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), M200 medium, D-glucose, Hoechst 33258 and mannitol were supplied by Sigma-Aldrich (St Louis, MO, USA). Cell counter kit-8 (CCK-8) was purchased from Dojindo Lab (Kumamoto, Japan). Anti-GAPDH (#8884), anti-ATF6 (#65880), anti-CHOP (#2895), anti-BiP (#3177), anti-phospho (p)-PI3K (#4228), anti-p-Akt (#4060), anti-p-eNOS (#9570), anti-total (t)-PI3K (#4249), anti-t-Akt (#4685), anti-t-eNOS (#9586), anti-Bax (#5023), anti-Bcl2 (#2827), anti-cleaved caspase 3 (#9661), anti-cleaved caspase 1 (#4199), anti-p-JNK(#4668), anti-t-JNK (#9252) and anti-gp91^phox (#80897) antibodies were from Cell Signaling Technology (Boston, MA, USA). Enhanced chemiluminescence (ECL) solution was purchased from KeyGen Biotech (Nanjing, China). Interleukin (IL)-1β (#ab46052), IL-6 (#ab46027) and tumor necrosis factor (TNF)-α (#ab10054) ELISA kits were provided by Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated secondary antibody and BCA protein assay kit were obtained from KangChen Bio-tech, Inc (Shanghai, China).

Three independent controls and miR-7-treated tumor samples were used for the protein arrays, according to the standard operating procedures of the Raybio (USA) human L493 array (catalog no. AAH-BLG-2-2). Total protein was extracted from 250 mg of tissues with ice-cold cell & tissue protein extraction reagent (KangChen Bio-tech, China, catalog no. KC-415), which contained the inhibitors for protein degradation (5 μL of protease inhibitor cocktail, 5 μL of PMSF, and 5 μL of phosphotase cocktail were added into 1 mL of protein extraction reagent). The protein concentration was determined by a BCA protein assay kit (KangChen Bio-tech, catalog no. KC-430). Protein array membranes were blocked in blocking buffer for 30 min and then incubated with samples at room temperature for 1–2 h, then decanted, and membranes were washed with washing buffer. After that, membranes were incubated with diluted biotin-conjugated antibodies at room temperature for 1–2 h. The membranes were washed with washing buffer and reacted with streptavidin-conjugated fluor at room temperature. Membranes were then washed thoroughly and scanned by an Axon scanner. The intensities of signals were quantified by densitometry. Raw intensities were revised by background and normalized by median. A 2-fold change in protein expression was calculated.25 (link), 26 , 27 (link)