Ccg 1423

CCG-1423, anti-NF-E2 related factor 2 (Nrf2), and anti-importin β1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Jasplakinolide (Jasp) and latrunculin B (LatB) were purchased ftom Caymen Chemical (Ann Arbor, MI). Other antibodies used in this study were anti-Flag M2 affinity gel (Sigma, St. Louis, MO), anti-HA affinity matrix and anti-HA (3F10) antibody (Roche Applied Science, Mannheim, Germany), and anti-DYKDDDDK (anti-Flag) antibody (Trans Genic, Kobe, Japan). Secondary antibodies and phalloidin were conjugated to Alexa 568 (Molecular Probes, Eugene, OR). We produced a polyclonal antibody against MRTF-A. In brief, a peptide consisting of amino acids 714–728 of mouse MRTF-A (MAL met; GenBank accession number: BC050941.1) was injected into a rabbit, and the antiserum was subjected to affinity purification.

The Protein BCA Assay kit (Beyotime, Jiangsu, China) was used to determine the total protein concentration of the cells and kidney cortex. The proteins were then separated by size using a 10% SDS-polyacrylamide gel electrophoresis followed by electrophoretic transfer of the resultant bands onto nitrocellulose membranes. The membranes were subsequently blocking with 5% skimmed milk powder supplemented with Tris-buffered saline-Tween buffer and then incubated overnight with primary antibodies at 4°C. The primary antibodies were MCP-1 (Santa Cruz Biotechnology, Inc.), β-actin (Cell Signaling Technology, CST, Boston, USA), ROCK1 (CST), GAPDH (CST), RhoA (CST), IL-6 (CST), PP65 (CST), CCG-1423 (Santa Cruz), and synaptopodin (Santa Cruz). The membranes were then incubated with the goat anti-rabbit/mouse immunoglobulin G horseradish peroxidase- (HRP-) conjugated secondary antibody (BioSharp, Technology Inc., China). The resultant banding patterns were analyzed using the Image Lab Software (Amersham imager 680, Cytiva, USA; UVP ChemStudio/PLUS, Analytik Jena AG, Germany).