Ta 10

Total protein was extracted from HK-2 cells using RIPA buffer (Sigma, R0278), and the protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, 23227). Next, denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and then electrically transferred to polyvinylidene difluoride membranes (Millipore, IPVH00010). The membranes were blocked for 60 minutes with 5% fat-free milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST). The blots were incubated with the following relevant primary antibodies overnight at 4°C: GSDMD (Abcam, ab209845, 1:1000), VCAM1 (Abcam, ab134047, 1:2000), and tubulin (ZSBio, TA-10, 1:5000). An incubation with a 1:1000 dilution of the HRP-conjugated secondary antibody was carried out for 1 hour at room temperature. After five washes with TBST, the membranes were incubated with the chemiluminescence substrate (Millipore, WBKLS0100) for 5 minutes, and images were captured using an Image Quant LAS 4000 Mini system (GE Healthcare). The semiquantitative analysis was conducted using ImageJ software (Media Cybernetics, Silver Spring, MD).

The separated proteins were electroblotted onto nitrocellulose filter membrane and blocked for 2 h at room temperature with Tris-buffered saline containing 5% BSA. Nitrocellulose filter membrane were incubated at 4 °C overnight with antibodies, rabbit anti-KLF7 (Abcam; ab197690), rabbit anti-IL-6 (Abcam, ab214429), rabbit anti-GPR120 (Abcam; ab230869), rabbit anti-GPR40 (Thermo Fisher; PA5-75351), rabbit anti-p-IKKβ (Cell Signaling Technology; 2697S), rabbit anti-T-IKKβ (Abcam; ab124957), rabbit anti-p-IκB (Cell Signaling Technology; 2859S), rabbit anti-T-IκB (Cell Signaling Technology; 4812S), rabbit anti-p-p65 (Cell Signaling Technology; 3033S), rabbit anti-T-p65 (Cell Signaling Technology; 8242S), rat anti-GAPDH (ZSGB-BIO; TA-08), rabbit anti-TBP (Cell Signaling Technology; 44059S), rat anti-β-Actin (ZSGB-BIO; TA-10) were used at a dilution of 1:1000. The secondary antibodies (ZSGB-BIO; ZB2301 and ZB2305) were incubated at 25 °C for 2 h at a working ratio of 1:10,000.

Cell culture media DMEM and RPMI-1640, fetal bovine serum (FBS) were ordered from Gibco. Human recombinant EGF (Catalog number:10605-HNAE) was bought from Sino Biological company. ML792 (HY-108702), SUMOylation inhibitor 2-D08 (HY-114166), gefitinib (HY-50895), puromycin (HY-B1743A) and N-ethylmaleimide (NEM) (HY-D0843), were ordered from MedChemexpress company. Slurry anti-Flag M2 affinity gel (A2220) was ordered from Sigma. The protein-A beads (161–4013) were bought from Bio-Rad. The primary antibodies included individual monoclonal antibody of Myc (ab18185, Abcam), EGFR (sc-373746, Santa Cruz), AnxA6 (sc-166807, Santa Cruz). And rabbit monoclonal antibodies against Flag (BX00086), HA (BX00069-C3), His (BX00085-C3), SUMO1(ET1606-53), UBC9(ET1610-21), pY-EGFR(1086) (ET1612-30) and p-ERK1/2 (ET1610-13), ERK1/2 (ET1601-29), Cyclin D1(SA38-08) were all ordered from HuaBio Company in China. The antibody of β-tubulin (TA-10, Zsbio) and β-actin (TA-09, Zsbio) was used to quantify expression of housekeeping gene β-tubulin or β-actin for comparison normalization. The IgG antibody (A7016, Beyotime) was taken as nonspecific binding control for IP performance.