Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page

Protein expressions of GPX1, PTEN, PDK-1, AKT, Bcl-2, Bax, and Nf-κB were detected by western blot using specific primary antibodies (Gene Tex, Irvine, California, US). Antibody against GAPDH (Kangcheng Biology, Shanghai, China) was used as a loading normalization control. After BAY 11-7082 treatment or transfection experiments, the cells were collected and the total protein was extracted from the cells. Protein (30 μg) was loaded and separated using a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Shanghai, China) gel and transferred to an Immobilon-P Transfer Membrane (PVDF) (Beyotime, Shanghai, China). The membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h at room temperature. The blots were probed with the selected primary antibodies overnight at 4°C, washed in TBST, and further incubated with a species-specific horseradish peroxidase-conjugated secondary antibody (anti-rabbit, ExCell Bio, Shanghai, China) for 1 hour at room temperature. The western blot process was performed according to standard protocols. An enhanced chemiluminescence detection method (Pierce ECL Western Blotting Substrate) (Thermo Scientific, Belmont, Massachusetts, US) was used to visualize the blots.

Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing proteinase and phosphatase inhibitors. A BCA assay kit (Beyotime) was used to measure the concentration of protein. The supernatant proteins were precipitated. The protein of lytic samples was transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, China) after separating by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime). Membranes were incubated with antibodies against gasdermin D (GSDMD, Abcam, Cambridge, UK), IL-1β (Abcam), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc-1, Abcam), TRAP (Abcam), pro caspase-1 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-1 (Cell Signaling Technology), ASC (Cell Signaling Technology), TNF receptor-associated factor (TRAF) 2 (Proteintech, Rosemont, IL, USA), TRAF6 (Proteintech), c-Fos (Proteintech), cathepsin K (CTSK, Proteintech), matrix metalloprotein 9 (MMP9, Proteintech), and anti-actin (Beyotime) overnight at 4 °C after blocking with quick block buffer (Beyotime) for 30 min. Then, membranes were washed three times with TBS-Tween and incubated with the appropriate secondary antibody for 1 h at room temperature. The results were visualised via chemiluminescent peroxidase substrate (Proteintech).

The total proteins in each group were extracted with a protein extraction kit (Camilo Biological, Nanjing, China). The protein concentration was quantified by bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA), and the protein was denatured by boiling for 10 min. 20 μg of the protein sample was electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Shanghai, China) and then transferred to polyvinylidene fluoride (PVDF) membranes (EpiZyme, Shanghai, China), followed by 5% bovine serum albumin (BSA) (Beyotime, Shanghai, China) to block nonspecific antigens of proteins. The membranes were then incubated with primary antibody (Bax, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; Bcl-2, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; DHCR24, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; PI3K, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; p-PI3K, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; AKT, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; p-AKT, Abcam, Cambridge, MA, USA, Rabbit, 1:1000; GAPDH, Abcam, Cambridge, MA, USA, Rabbit, 1:1000) at 4°C overnight, and then incubated with secondary antibody. Finally, the gel imaging system electrochemiluminescence (ECL) was used for development.

TCDCA (purity ≥ 98%) and TLCA (purity ≥ 97%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). INT-777 (purity ≥ 98%) was purchased from MedchemExpress Co. (Princeton, NJ, USA). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was purchased from Beyotime Co. (Beijing, China). pMD19-T vector, Hind restriction endonuclease III (Hind III), restriction endonuclease XbaI, and SYBR^® Premix Ex Taq^TM were purchased from Takara Co. (Shiga, Japan). pCMV-C-EGFP plasmid and pCMV-Blank plasmid were purchased from Tiangen Co. (Beijing, China). Lipofectamine^TM 2000 transfection reagent was purchased from Invitrogen Co. (Carlsbad, CA, USA). Sheep anti-rabbit TGR5 antibody, Rabbit anti-goat TGR5 antibody, and Rhodamine-labeled mouse anti-goat antibody were purchased from Cruz Santa Co. (Dallas, TX, USA). Horseradish peroxidase-labeled Goat anti-rabbit antibody was purchased from KPL Co. (Gaithersburg, MD, USA). cAMP ELISA Kit was purchased from Cayman Co. (Ann Arbor, MI, USA). The Bright-Glo^TM Luciferase Assay Kit was purchased from Promega Co. (Madison, WI, USA). The Nuclear Extract Kit was purchased from Active Motif Co. (Carlsbad, CA, USA). Dulbecco’s Modified Eagle Medium was purchased from Hyclone Co. (Shanghai, China). Fetal bovine serum was purchased from ExCell Co. (Taicang, China). Other reagents were purchased from ExCell Co.