Ha1024

The total protein of HHNs was extracted using a total protein extraction kit (#BB-3101; BestBio, Shanghai, China) and the concentration of isolated total protein was measured using a BCA protein quantification kit (#BB-3401; BestBio). Next, equivalent amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE, #YJ0014B; Yiji, Shanghai, China) and transferred onto a polyvinylidene difluoride (PVDF, #SD7966555; Yiji) membrane. Subsequently, the PVDF membrane was blocked with 5% skim milk at room temperature for 1 h, then treated with primary antibodies against NF-κB (1:1,000, #ER0815; HUABIO, Hangzhou, China), anti-NLRP3 (1:1,000, #ER1706-72; HUABIO), anti-caspase-1 (1:1,000, #ET1608-69; HUABIO), anti-IL-1β (1:1,000, #ET1701-39; HUABIO), anti-IL-18 (1:1,000, #EM170401; HUABIO), anti-TIGAR (1:1,000, #ER62495; HUABIO), and anti-GAPDH (1:1,000, #ER1901-65; HUABIO) overnight at 4°C. The next day, a secondary antibody (1:1,000, #HA1024; HUABIO) was used to treat the PVDF membrane at 37°C for 1 h. Protein bands were visualized using an ECL chemiluminescence detection kit (#BB-3501; BestBio). ImageJ was used to analyze the gray values of bands.

According to the instructions, PMSF and RIPA tissue / cell lysates (Solarbio, China) were added to cells or tissues, lysed on ice for 20 min, and then centrifuged for 15 min (4 ° C, 12,000 rpm). Then, the protein concentration of the supernatant was measured using the BCA concentration kit (Beyotime, China). Finally, SDS-PAGE protein loading buffer (5X) (Beyotime, China) was added, and then the protein was denatured by metal bath for 10 min. After the protein extraction was completed and the concentration was determined, an appropriate amount of sample protein was added to the electrophoresis gel for electrophoresis separation, and then transferred to the PVDF membrane (Solarbio, China). PVDF membranes were sealed with a rapid blocking solution and incubated overnight with primary antibody in a refrigerator at 4 ° C, and the next day with secondary antibody (1:20000, HA1024; HUABIO) were incubated at room temperature for 60 min. In the last step, we used the Ori Ultrasensitive ECL Kit (Oriscience, China) to observe the bands. The antibodies used were OLFM2 (1:1000, ab154196; Abcam), GAPDH (1:30000, SA30-01; HUABIO), E-cadherin (1:5000, ET1607-75; HUABIO), N-cadherin (1:1000,ET1607-37; HUABIO), Vimentin (1:20000,ET1610-39; HUABIO), TGF-βR1 (1:1000,PB1154; BOSTER), Phospho-Smad3 (1:1000,C25A9; CST), Phospho-Smad2 (1:1000, D27F4; CST).