Bca protein assay kit

Cells were lysed in RIPA buffer, and protein concentrations were determined by a BCA Protein Assay Kit (Santa Cruz, CA, USA). Membrane protein was purified by Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Scientific™, MA, USA). A total of 30-50 μg of protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Immunoreactions were detected by an imaging system (Bio-Rad, USA).

Cells were lysed in RIPA buffer and protein concentrations were determined by a BCA Protein Assay Kit (Santa Cruz, CA, USA). Immunoblotting were performed as described previously [22 (link)]. For immunoprecipitation, extracts were pre-cleared with 30 μL agarose A/G beads (Beyotime Institute of Biotechnology) by rotating for 1 h at 4°C. Beads were removed, and then another 30 μL agarose A/G beads and 1.5 μg antibodies were added to the lysates rotating overnight at 4°C. The beads were then washed twice in basic lysis buffer, and boiled for 10 min in loading buffer before resolving by SDS-PAGE. The blots were detected by an imaging system (Bio-Rad, USA).

Cells were lysed in RIPA buffer and protein concentrations were determined using a BCA Protein Assay kit (Santa Cruz Biotechnology, Santa Cruz, CA). Equal protein was assayed by immunoblot using anti-rabbit HMGB1 (1:1000, Abcam, Cambridge, UK), anti-rabbit p21 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse p53 (1:1000, Cell Signaling Technology, Danvers, MA), anti-rabbit Sp1 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), or anti-β-actin (1:10000, Sigma-Aldrich Corp., St. Louis, MO) antibodies followed by peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, Sigma-Aldrich Corp., St. Louis, MO).

Inner sections of kidney poles were minced and homogenized in ice-cold RIPA buffer with protease inhibitors (0.15 M NaCl, 50 mM Tris-HCl pH 8.0, 1% NP-40, 0.5% deoxycholate). Total protein content was determined using the BCA Protein Assay Kit (Santa Cruz, CA, USA) according to manufacturer instructions. Protein samples (40 micrograms) were separated on a precast NuPAGE 10% Bis-Tris gel (Novex) at 200 v for 45 min. Blots were blocked at 25 °C and incubated with primary antibodies. Then, the membranes were incubated with the secondary antibodies (1:1000 dilutions) and analyzed by normalization against β-actin bands (used as a housekeeping gene). Fibronectin, TGF-β1, and NOX-4 protein levels were detected using polyclonal rabbit antibody from Santa Cruz, CA, used at 1:200 dilutions. Immunoblots are shown in each figure as representative images. Results are shown as the ratio of each band vs. β-actin (fold change of control). Analysis was conducted by using 4–6 animals per group and 3–5 independent experiments for Western blot analysis (Supplementary Figures S1 and S2). Blots were incubated with chemiluminescent substrate ECL reagent (Perkin Elmer, Waltham, MA, USA) by direct exposure on films that were scanned and analyzed by ImageJ software.

Protein lysates of cell samples or NP tissues were prepared by RIPA lysis buffer plus 1% phenylmethanesulfonyl fluoride. The total protein concentrations were measured with a BCA protein assay kit (Santa Cruz). Proteins were then separated by 10% SDS‐PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were then blocked by 5% bovine serum albumin for 2 hours at room temperature, followed by incubation with primary antibodies at 4°C overnight. After that, the membranes were incubated with second antibodies (1:5000, Abcam, ab150077) at room temperature for 2 hours. Protein bands were then detected using an enhanced chemiluminescence kit (ThermoFisher) and quantified by Image J software. Antibodies against NLRP3 (1:2000, ab214185), IL‐18 (1:1000,ab71495), IL‐1β (1:2000, ab234437), Gasdermin D (GSDMD, 1:1000, ab219800), TSG101 (1:2000, ab125011), and GAPDH (1:5000, ab181602) were purchased from Abcam. Antibodies against CD9 (1:2000, #13403), CD81 (1:2000, #56039), CD63 (1:2000, #55051), GM130 (1:1000, #12480), Cleaved caspase‐1 (1:1000, #89332), cleaved GSDMD (1:1000, #50928) and Calnexin (1:1000, #2679) were purchased from CST.

Protein expression levels were assessed through Western blot analysis. Proteins were extracted from the U87 and U251 cell lines utilizing RIPA lysis buffer, and their concentrations were subsequently semi-quantified using a BCA protein assay kit provided by Santa Cruz Biotechnology (Dallas, TX, USA). Proteins, in the amount of 20 µg per lane, were resolved via 12% SDS-PAGE and subsequently transferred onto PVDF membranes. These membranes were then blocked using 5% BSA sourced from Sigma-Aldrich for 1 h at ambient temperature before the blocking solution was discarded. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies: anti-GPR27 (PA5-110977, Invitrogen, Cambridge, MA, USA) and anti-GAPDH (MA1-16757; Invitrogen, Cambridge, MA, USA), both diluted at a ratio of 1:2000. Following the primary antibody incubation, the membranes were washed with TBST containing 0.1% Tween. Then, an HRP-conjugated secondary antibody was applied to incubate at room temperature for 1 h. Detection of the proteins of interest was achieved using the ECL Western Blotting substrate kit (Cattegory #ab65623; Abcam). Each experiment was repeated three times independently.