Anti fibronectin ab2413

The protein concentration was measured using a Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific). Approximately 10 μg of protein from each cell was subjected to 8% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membranes were blocked with 3% bovine serum albumin solution in TBST, followed by 1 h incubation. After that, the membrane was incubated with primary antibody at 4 °C overnight. The primary antibodies used to probe each protein were as follows: mouse anti-phosphoSmad2/3 (8828, Cell Signaling, Danvers, MA, USA): 1:1000, rabbit anti-Smad2/3 (8685, Cell Signaling): 1:1000, anti-fibronectin (ab2413, Abcam): 1:1000, mouse anti-β-actin (4967, Cell Signaling): 1:1000, mouse anti-phospho-histone H2A.X (05-636, Merck): 1:1000, rabbit anti-cleaved caspase3 (9664, Cell Signaling): 1:1000, rabbit anti-caspase3 (9662, Cell Signaling): 1:1000, rabbit anti-p62 (610832, BD Bioscience, Franklin Lakes, NJ, USA): 1:500 and rabbit anti-LC3 (NB100-2331, Novus Biologicals, Centennial, CO, USA): 1:1000. Secondary horseradish peroxidase (HRP)-conjugated antibodies (G21040, G21234, Invitrogen): 1:2000. The antibody binding was detected using an enhanced chemiluminescence (ECL) detection kit (RPN2106, GE Healthcare Life Science).

Anti-cleaved PARP (#9541), anti-LC3 (#12741) anti-p-cdc2 (#4139), anti-cyclin B (#4138), anti-vimentin (#3932), and anti-β-actin (#3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (ab2413) was purchased from Abcam (Cambridge, UK). Sorafenib tosylate (marketed as Nexavar by Bayer, Leverkusen, Germany) was purchased from Selleckchem (Houston, TX, US). For in vitro experiments, sorafenib was dissolved in dimethyl sulfoxide to generate a 20 mmol/L stock solution, which was stored at 4 °C until use.

The aristolochic acid I sodium salt used to generate mouse model of acute AAN was purchased from Sigma (St. Louis, MO, USA). The recombinant mouse IL-22 was obtained from Novoprotein (Shanghai, China) and the recombinant human IL-1Ra was kindly provided by General Regeneratives Limited (Shanghai, China). The primary and secondary antibodies used for immunoblot analysis were as follows: anti-Fibronectin (ab2413) and anti-Collagen IV (ab6586) from Abcam (Cambridge, Massachusetts, USA); anti-α-Smooth Muscle Actin (α-SMA) (14968), anti-Vimentin (D21H3) (5741), anti-NLRP3 (15101), anti-Cleaved Caspasse-1 (67314), anti-IL-1β (12242), and anti-β-actin (8H10D10) (3700) from Cell Signaling Technology (Danvers, MA, USA); peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G (IgG) from Jackson ImmunoResearch Laboratory (West Grove, PA, USA). The primary and secondary antibodies used for immunohistochemical staining of NLRP3 were as follows: anti-NLRP3 (GB11300) and HRP-conjugated goat anti-rabbit IgG (GB23303) from Servicebio Technology Co. Ltd. (Wuhan, China).