Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page

The CD34+CD38− LSCs cells were lysed using protein lysis buffer (Applygen Tech. Inc., Beijing, China). The protein concentration of the lysates was evaluated using the bicinchoninic acid (BCA) protein concentration detection kit (Cat. No. P0012) (Beyotime Biotech., Shanghai, China). The lysates were separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma-Aldrich, St. Louis, MO, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Beyotime Biotech., Shanghai, China). The PVDF membranes were incubated overnight at 4°C in the primary antibodies, which were all purchased from Abcam Biotech (Cambridge, MA, USA). The primary antibodies included rabbit anti-human SIRT1 monoclonal antibody (Cat. No. ab32441) (1: 2000), rabbit anti-human TSC2 monoclonal antibody (Cat. No: ab190349) (1: 2000), rabbit anti-human polyclonal antibody (Cat. No. ab8227) (1: 2000). The membranes then incubated in the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat. No. ab6721) (1: 1000) (Abcam Biotech., Cambridge, MA, USA) at 37°C for 1 h. Western blot images were visualized using the electrochemiluminescence (ECL) kit (Pierce Thermo Scientific, Rockford, IL, USA).

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).