Horseradish peroxidase hrp conjugated secondary antibody

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).

Orbital fibroblasts (5×10⁵/well) were seeded in six-well plates, and the proteins from the cells were extracted using RIPA lysis buffer (Beyotime, Nantong, China). For western blotting, equal amounts of proteins were separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted onto a prewetted nitrocellulose membrane (GE Healthcare, Munich, Germany), followed by blocking of the membranes in 10% defatted milk in 0.2 M phosphate buffer saline (PBS; 1X; 32 mM NaH₂PO₄, 168 mM Na₂HPO₄, pH=7.5) at 4 °C for 4 h. The membranes were then probed with different primary antibodies. The following primary antibodies were used: mouse anti-ki67 (1:1,000), anti-Bcl-2 (1:1,000), anti-Bax (1:1,000), anti-α-SMA (1:1,000), anti-p-Smad3 (1:1,000), anti-Smad3 (1:1,000), and anti-β-actin monoclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA). After extensive washing with Tris Buffered Saline Tween-20 (TBST; 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH=7.5 ), the membranes were incubated for 1 h at 25 °C with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000; KangChen Bio-tech, Shanghai, China). Specific bands were visualized using enhanced chemiluminescence (ECL) reagent (Beyotime). Luminance was scanned using a Typhoon scanner (Amersham Biosciences, Piscataway, NJ). All experiments were performed in triplicate.