Ecl western blotting system

Proteins were extracted using RIPA buffer, and protein concentration was measured using BCA Kit (EnoGene, Nanjing, China). Proteins were separated by 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane (Bio-Rad, USA). The membranes were blocked with 5 % skimmed milk at room temperature for 2 h, and washed in TBS-Tween 20. Subsequently, the membranes were incubated with anti-ROR2 (Biovision, Cat. 6702-100), anti-Bax (EnoGene, Cat. E11-0132C), anti-Bak (EnoGene, Cat. E11-0131C), anti-Bcl-2 (EnoGene, Cat. E10-30077), anti-Bcl-xl (EnoGene, Cat. E90209), anti-mTOR (EnoGene, Cat. E11-7156B), anti-survivin 1 (Biorbyt, Cat. orb394299), anti-PI3K (Biorbyt Cat. orb137259), anti-AKT (bioss, Cat. bs-0115R-1), anti-pAKT (bioss, Cat. bs-12458R-1), anti-PDK1 (EnoGene, Cat. E10-30154), anti-p21 (Abcam, Cat. ab215971-p21), anti-Cyclin D1 (Abcam, Cat. ab185241 - Cyclin D1), and control anti-GAPDH (EnoGene, Cat. E12-052) antibodies at 4 °C overnight. After washing in TBS-Tween 20, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP, EnoGene) at 37 °C for 1 h. Protein bands were detected using ECL Western Blotting System (Millipore, MA, U.S.A.) and visualized using image analyzer (DKSH, USA). The immunoblot signal was quantitated with ImageJ software and the values were normalized to the GAPDH band density.

After washing with precooled PBS, proteins from cells and tissues were lysed and extracted by radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, USA) mixed with protease inhibitors. The mixtures were centrifuged at 12,000 × g for 15 min to collect the supernatant as the protein solution. BCA protein assay kits were used to determine the protein concentration in the solutions. For the western blotting analysis, the protein solution was boiled at 100 °C for 10 min after evenly mixing with 5 × loading buffer. Denatured protein solutions were prepared as previously described [25 (link)]. The membranes were incubated with the primary antibodies against TLR4 (1:200, sc-293072, Santa Cruz, USA), TRAF6 (1:1000, 67591, CST, USA), IRAK1 (1:1000, 4504, CST, USA), NF-κB (1:1000, 4764, CST, USA), phospho-NK-κB (1:1000, 3033, CST, USA), β-Actin (1:1000, AF0003, Beyotime, China) at 4 °C for 12 h, followed by incubation with the secondary antibodies at room temperature for 1 h. Then, an enhanced chemiluminescence system (ECL Western Blotting System) (Millipore, USA) was used to visualize the immunoreactive proteins on the membranes. The protein bands were quantified and analyzed with ImageJ 1.34s software.

Cells were lyzed in 2.5× sodium dodecyl sulfate (SDS) gel loading buffer (30 mM Tris-HCl, pH 6.8, 1% SDS, 0.05% bromphenol blue, 12.5% glycerol, and 2.5% mercaptoethanol) and boiled for 30 min; the proteins were prepared for separation on 12% SDS polyacrylamide gels, followed by electro-transferring to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) and incubated with the primary antibodies: rabbit anti-Bcl-2 (1:1,000, ab32124,Abcam, United Kingdom), rabbit anti-Bax (1:1,000, ab32503, Abcam, United Kingdom), rabbit anti-cleaved caspase3 (1:1,000, ab49822, Abcam, United Kingdom), rabbit anti-cytochrome c (1:1,000, 4272s, CST, MA, USA),rabbit anti-JNK1 + JNK2 + JNK3 (phosphor T183 + T183 + T221) (1:1000, ab124956,Abcam, United Kingdom), rabbit anti-JNK1 + JNK2 + JNK3 (1:1,000, ab179461,Abcam, United Kingdom), rabbit anti-Keap1 (1:1,000, ab139729, Abcam, United Kingdom), Nrf2 (1:1,000, ab31163,Abcam) and mouse anti-β-actin (1:3,000, KC-5A08, Kangcheng, China). Protein bands were visualized after horseradish peroxidase-conjugated secondary antibodies incubation and detected by a chemiluminescence ECL Western-blotting system (Millipore, MA, USA).

Cells were rinsed with cold PBS, lysed in 5× SDS gel loading buffer, and boiled for 10 min; then, the proteins in the lysate were separated on 12% SDS polyacrylamide gels, electrotransferred to polyvinylidene difluoride membranes (Millipore, Boston), and stained with the following primary antibodies: GSDMD (1:1,000; Abcam, ab219800), GSDME (1:1,000; Abcam, ab215191), cleaved caspase-3 (1:1,000; Cell Signaling Technology, #9664), and β-actin (1:1,000; Abcam, ab8226). Primary antibodies were stained with horseradish-peroxidase-conjugated secondary antibodies (1:10,000; Abcam, ab205718) and visualized with a chemiluminescence ECL Western blotting system (Millipore)