Anti mcp 1

Tissue samples from the renal tissue were placed in a buffer containing 20 mM Tris-HCl, pH 6.8, 1 mM EDTA, 1% SDS, 1 mM PMSF and 1× protease inhibitor cocktail. The protein was separated on 15% SDS-PAGE and electroblotted onto nitrocellulose (NC) membranes. The membranes were incubated with one of the following antibodies: anti- p65 (1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-p-Akt (Ser⁴⁷³,1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-α-SMA(1:1000; Abcam, USA); anti-MCP-1(1:200; Santa Cruz Biotechnology. Santa Cruz, CA, USA); anti-TGF-β1(1:500; Santa Cruz Biotechnology. Santa Cruz, CA, USA);as the primary antibody. HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody (1:1000; Sigma, USA). All membranes were incubated with a monoclonal anti-β-actin antibody (1:2000; Novus, USA). Immunoreactive bands were visualized with the luminescence method (Western Blot Chemiluminescence Reagent Plus, NEN™ Life Science Products Inc.). The band density was normalized to the corresponding density of β-actin at 42 kDa.

For Western blotting, the lysates from tissue and treated cells were prepared, then separated by SDS-PAGE, and transferred to PVDF membranes. The following primary Abs used for western blot analysis: anti-IL-33 (Abcam, Cambridge, UK), anti-ST2 (Abcam, Cambridge, UK), anti-IL-1β (Cell Signaling Technology, Danvers, MA, USA), anti-MCP-1 (Santa Cruz Biotechnology, California, USA), anti-STAT3 (Cell Signaling Technology, Danvers, MA, USA), anti-pSTAT3 (Cell Signaling Technology, Danvers, MA, USA), anti-NF-κBp65 (eBioscience) and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA). Immune reaction bands were detected by using horseradish peroxidase-labeled, species-specific secondary antibodies (Cell Signaling Technology) and enhanced chemiluminescence analysis (EMD Millipore, Billerica, MA, USA), and viewed by Kodak Image Station 4000MM (Eastman Kodak, Rochester, NY, USA).

The kidney cortical tissues were homogenized in RIPA lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS). Protein concentration was determined by Bradford method. Aliquots of 40–60 μg of protein were separated by SDS-PAGE (10% resolving gel), transferred to PVDF membranes (Millipore, Boston, MA, USA), and blocked with 10% nonfat milk. Membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were applied: anti-nephrin (1:400, Abcan, Cambridge, UK), anti-podocin (1:400, Abcan, Cambridge, UK), anti-TNF-α (1:300, Santa Cruz, CA, USA), and anti-MCP-1 (1:300, Santa Cruz, CA, USA). Primary antibodies were detected by secondary antibodies (1:2000, Santa Cruz, CA, USA). The protein bands were visualized with chemiluminescence reagent (ECL, Cell Signaling, Beverly, MA, USA) and quantified using Scion Image software. After determination of target proteins, membranes were incubated in stripping buffer (0.2 mmol/L glycine, 0.02% Tween 20, PH 7.5; 20 min at 80 °C), washed extensively, and subjected to immunoblotting analysis to determine β-actin (1:2500, Santa Cruz, CA, USA).

Brain tissues, cells or exosomes were harvested at the indicated times. Protein lysates were prepared with RIPA buffer (Beyotime) containing protease inhibitor cocktail (Thermo). BCA protein assay kit (Beyotime) was used for protein concentration detection. Proteins were removed onto PVDF membrane (Millipore) after isolated in SDS-PAGE (10%). 5% non-fat milk was applied to block the membrane for 1 h. Then the membrane was incubated in primary antibodies overnight at 4 °C. Primary antibodies anti-IL-6, anti-TNFα and anti-MCP-1 were acquired from Santa Cruz, while anti-Sirt1, anti-USP22, anti-MDM2, anti-p-JNK and anti-β-actin were obtained from Abcam. Following incubating in secondary antibodies conjugated with HRP (Thermo), the blots were then visualized by ECL reagent (Beyotime).