Clarity ecl substrate kit

CRC tissues and cells were treated with lysis buffer (Beyotime) to prepare protein samples. Protein concentration was measured using bicinchoninic acid assay (BCA) kit (Beyotime). And 20 μg protein was loaded on 12% bis-tris-acrylamide gel (Thermo Fisher). Then, protein bands were transferred onto polyvinylidene fluoride (Millipore) and immersed in 5% nonfat milk (Solarbio, Beijing, China) at 4 °C for 4 h. After incubated with anti-B cell lymphoma-2 (anti-Bcl-2) (1:1000; CST, Boston, MA, USA), anti-BCL2-associated x protein (anti-Bax) (1:1000; CST), anti-GRIK3 (1:1000; Abcam, Cambridge, UK), and anti-β-Actin (1:1000; CST) at 4 °C overnight, the membranes were incubated with the secondary antibody labeled with horseradish peroxidase (1:2000; CST) at 37 °C for 2 h. The protein bands were developed using a Clarity™ ECL Substrate Kit (Bio-Rad, Shanghai, China). β-Actin was chosen as a control.

The Western blot procedure was carried out as previously described [25 (link)]. In short, 40 μg protein lysate underwent separation using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then was transferred onto a PVDF membrane. The membranes were then blocked for one hour using 5% skimmed milk. Then, the blots were exposed to primary antibodies against INS (Cat. #8138; Cell Signaling Technology, Danvers, MA, USA), INSRβ (Cat. #ab69508; Abcam), PDX1 (Cat. #5679, Cell Signaling Technology), GLUT2 (Cat. #A12307; ABclonal, Woburn, MA, USA), NEUROD1 (Cat. #A1147; ABclonal), MAFA (Cat. #ab264418), GCK (Cat. #ab37796), VAMP2 (Cat. #13508), and SNAP25 (Cat. #MA5-17609; Thermofisher, Waltham, MA, USA) overnight at 4 °C. The β-actin (Cat. #A5441; Sigma, St. Louis, MO, USA) was used as an endogenous control. Following the wash with TBST, the blots were then incubated with secondary antibodies, HRP-linked anti-mouse (#7076S, Cell Signaling, Danvers, MA, USA), and HRP-linked anti-rabbit (#7074; Cell Signaling) for 1 h at room temperature. Chemiluminescence was recorded using the Clarity ECL substrate kit and ChemiDoc Touch Gel Western Blot Imaging System (Bio-Rad, Hercules, CA, USA). The protein bands were quantified using the Bio-Rad Image Lab software (6.1).

To detect activated inflammasome proteins, INS-1 cells were stimulated with 1 µL LPS/200 μM PA–BSA for 4 h [24 (link),59 (link)]. Total protein extraction was performed using ice-cold NP-40 (1.0% NP-40, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0) lysis buffer containing a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). A Western blot analysis was performed as previously described [58 (link)] with the following antibodies: MAPK8IP1 (anti-rabbit; 1:1000, #Ab24449, Abcam, Cambridge, UK), NLRP3 (anti-rabbit; 1:1000, #A12694, Abclonal, Woburn, MA, USA), CASPASE-1 (anti-rabbit; 1:1000, #A0964, Abclonal, Woburn, MA, USA), IL-1β (anti-rabbit; 1:1000, #A162888, Abclonal, USA), GSDMD (anti-rabbit; 1:1000, #A10164, Abclonal, USA), JNK (anti-rabbit; 1:1000, #A48567, Abclonal, USA), pJNK (anti-rabbit; 1:1000, #AP0631, Abclonal, USA), β-actin (anti-mouse, 1:1000, #A5441, Sigma-Aldrich, Darmstadt, Germany), and secondary anti-mouse (#7076S) or anti-rabbit (#7074S, from Cell Signaling Technology, Danvers, MA, USA). Chemiluminescence was detected using the Clarity ECL substrate kit (Bio-Rad, Hercules, CA, USA). β-actin was used as an endogenous control.