Loading buffer

After centrifugation, cell pellets from each of the previously mentioned cell groups were collected. After adding the cell lysate RIPA (Fudebio, Hangzhou, China), cells were placed on ice for 30 min and then centrifuged at 12,000 r·min⁻¹ for 4 min at 4 °C. Next, the supernatant was collected, and protein in the supernatant was assessed with the BCA method (Fudebio, Hangzhou, China). 50 μg of this protein was added to 5 × loading buffer (Fudebio, Hangzhou, China) at a volume ratio of 4:1, and boiled at 100 °C for 5 min to prepare a protein sample. After loading, the protein was subjected to vertical electrophoresis at 80 V for 30 min, which was then continued with 120 V until the bromophenol blue ran out of the glass plate. Next, the protein gel was removed, and the membrane was incubated at 100 V in an ice bath, while 5% of skim milk powder was incubated for 6 h at room temperature. Then, the corresponding primary antibodies (1:1000) for Bax, Bcl-2, P65, p-P65, p-IKBα, IL-17, and GAPDH (Affinity biosciences OH, USA) were added to shake at 4 °C overnight. The sample was then washed with TBST washing solution, and horseradish peroxide was added. Next, the enzyme-labeled secondary antibody (1:5000) was incubated at 37 °C for 1 h. After washing with TBST, it developed with an ECL illuminant (Fudebio, Hangzhou, China), and images were acquired using an automated gel imaging system.

Silver staining was performed using a Fast Silver Stain Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol. Briefly, the proteins obtained from the ChIRP assay denatured at 95 °C in 5 × loading buffer (Fudebio, Zhejiang, China) for 5 min and loaded onto SDS-polyacrylamide gel for electrophoresis. After fixing with a stationary liquid (50 mL ethanol, 10 mL acetic acid, and 40 mL double-distilled H₂O) for 1 h, the gel was washed with 30% ethanol for 10 min and twice with double-distilled H₂O. The gel was incubated with a silver staining buffer for 10 min and washed twice with double-distilled H₂O. Gels were stained with a chromogenic agent until clear bands were visible, after which they were washed with the corresponding elimination agent.