Anti p65

The antibodies, reagents and plasmids used in this study were as follows: X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, Grenzach, Germany); anti-CHAC2 (Proteintech, Rosemont, IL, USA); anti-p65 (Affinity Biosciences, Changzhou, China); anti-pp65 (Affinity Biosciences, Changzhou, China); anti-GAPDH (Cell Signaling Technology (CST), Danvers, MA, USA); anti-NOD1 and anti-TLR4 (Affinity Biosciences, Changzhou, China), ML130 inhibitor and TAK-242 (Selleck, Houston, TX, USA), pcDNA3.1 plasmids (Invitrogen, Carlsbad, CA, USA).

The kidney tissue sections (5 μm) were deparaffinized, rehydrated, and washed with PBS. Cells were fixed in 4% paraformaldehyde, incubated with 0.1% Triton X-100 (Beyotime), and washed with PBS. Tissue sections and cells were blocked in goat serum for 15 min. For TOLLIP-TLR2 and TOLLIP-TLR4 staining, tissue sections were subjected to incubation with anti-TOLLIP (ABclonal, China) and anti-TLR2 (NOVUS, USA)/anti-TLR4 (Santa Cruz, USA) antibodies (1 : 50 dilution) at 4°C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG or Cy3-conjugated goat anti-mouse IgG (Beyotime; 1 : 200 dilution) at room temperature for 90 min. For cleaved caspase-3, NLRP3, and p65 staining, tissue sections or cells were subjected to incubation with anti-cleaved caspase-3 (Affinity, China), anti-NLRP3 (ABclonal), and anti-p65 (Affinity) antibodies (1 : 100 dilution) at 4°C overnight, followed by incubation with Cy3-conjugated goat anti-rabbit IgG (1 : 200 dilution) at room temperature for 60 min. Next, tissue sections or cells were subjected to dihydrochloride (DAPI) (Aladdin) nuclear staining. Images were captured using a fluorescence microscope (OLYMPUS).

Western blotting was performed as described previously [54 (link)]. Briefly, cells and liver tissue samples were collected and lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Solarbio, Beijing, China). Protein concentration was measured using a BCA protein assay kit (LABLEAD, Beijing, China). Proteins were separated by 10% SDS-PAGE gel at 115 V for 1.2 h, then were transferred to a PVDF membrane at 200 mA for 1 h. The membranes were blocked with 5% milk in TBST for 1 h and incubated overnight at 4 °C with primary antibodies. Primary antibodies included anti-Smad4, anti-α-SMA, anti-GAPDH, anti-E-cadherin, anti-ID1, anti-CTGF, anti-p65, anti-p-p65, anti-p38, and anti-p-p38 (Affinity Biosciences, Cincinnati, OH, USA). Followed by HRP-conjugated goat, anti-mouse and goat anti-rabbit IgG (Solarbio, Beijing, China) were used as secondary antibodies. Protein bands were scanned using a Clinx Science Instrument and quantified with Image J software.