Radioimmunoprecipitation assay (ripa)

Details of Western blotting were previously described^{18 (link)}. Cells at 80–90% confluence were lysed on ice in radioimmunoprecipitation assay buffer (RIPA; keygen biotech, China) containing PMFS complete protease inhibitor cocktail (keygen biotech, China). Protein concentration was determined by the BCA assay (keygen biotech, China). Equal protein samples (10 μg) were separated on 12% sodium dodecyl sulfate (SDS)/polyacrylamide gels, and transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA). The antibodies used were as follow: anti-IMPA2 rabbit monoclonal antibody (1:1000; GeneCopoeis, USA); anti-ERK (1:10,000, Abcam, UK), anti-p38α (1:500, BBI China), anti-JNK1/2/3 (1:500, BBI China), p-ERK (1:500, Cell Signaling, USA), p-p38α (p-Thr180/Tyr182, 1:500, BBI China), and p-JNK1/2/3 (p-Th183/Ty185, 1:500, BBI China). Horseradish peroxidase (HRP)-conjugated goat anti‑rabbit immunoglobulin G (1:1000; BBI China) was used as second antibody and anti-GAPDH mouse monoclonal antibody (1:5000; BBI China) as a loading control. The final protein expression was detected by enhanced chemiluminescence (Bio-rad, Berkeley, CA, USA) according to the manufacturer’s suggested protocols. The band quantification was conducted using ImageJ (National Institutes of Health, Bethesda, MA, USA).

About 1 × 10⁶ Beas 2B cells were put in each well and lysed with 80 µL RIPA (KeyGen) buffer for 20 min at 4℃ and oscillated by vortex every 5 min. The bicinchoninic acid protein assay kit (Beyotime) was used to estimate the concentration of protein. Each sample was loaded with 30 µg of protein and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore) and blocked in buffer at 37°C for 1 hour. Primary antibodies (SIRT1, 1:1,000, Abcam; NLRP3, 1:800, Novus; ASC, 1:1,000, Abcam; and caspase-1, 1:1,000, Abcam) were diluted and incubated overnight at 4°C. The membranes were then incubated with the secondary antibody at room temperature for an hour. Chemiluminescence imaging was used to visualize the membranes.

Lysis buffer (RIPA, KeyGEN) containing protease inhibitors (PMSF, KeyGEN) was used to extract protein of cells and tissues, and protein concentration was detected with a BCA Kit (KeyGEN). Protein samples (40 μg) were loaded into 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDSPAGE) gels and transferred onto a PVDF membrane after electrophoresis. The membrane was blocked with non-fat milk for 2 h, and incubated overnight with antibodies against respective antibodies: MNX1 (Abcam, ab79541, 1:1,000); p21^cip1 (Cell Signaling Technology; 2947, 1:1,000); p^Thr161-CDK1 (Cell Signaling Technology, 9114, 1:1,000); CDK1 (Cell Signaling Technology, 9116, 1:1,000); p27^kip1 (Cell Signaling Technology, 3686; 1:1,000); CyclinB1 (Abcam, ab72, 1:1,000); CyclinE1 (Abcam, ab3927, 1:1,000); CyclinE1 (Abcam, ab3927, 1:1,000); CyclinD1(Santa Cruz Biotechnology, sc-246, 1:1,000); β-actin (Abcam, ab15265, 1:1,000).

Western blot analysis was performed as previously described [3 (link)]. In brief, PCa cells were added in radio immunoprecipitation assay buffer (RIPA, KeyGEN, BioTECH, China), and BCA Protein Assay Kit (KeyGEN, BioTECH, China) was used for protein determination. Then, the proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in no fat milk and then incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-Tubulin antibody (Cell Signaling Technology, USA, 1:1000), anti-ARHGAP5 antibody (ABclonal, China, 1:1000), anti-HDAC4 antibody (ABclonal, China, 1:1000), anti-IGF2BP3 antibody (Abcam, USA, 1:1000), anti-ZEB-1 antibody (Cell Signaling Technology, USA, 1:1000), anti-E-Cadherin antibody (Cell Signaling Technology, USA, 1:1000), anti-Vimentin antibody (Cell Signaling Technology, USA, 1:1000), and phospho-Erk1/2-T202/Y204 antibody (ABclonal, China, 1:1000). Subsequently, the membranes were immersed with the secondary antibody for 1 h. Finally, enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, USA) was used to detect the level of protein.