Anti klf4 antibody

Eight confluent 10-cm dishes of PDGFR-β⁺ cells or SMCs (~8 × 10⁶ cells) were treated with 1% formaldehyde for 10 min to cross-link protein to DNA and were neutralized with glycine. Cells were scraped in cold PBS and subjected to ChIP assay using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling) with an antibody targeting KLF4 (Abcam) or Histone H3K9Ac (Cell Signaling). Coprecipitated DNA was analyzed by qPCR with primer sets (sense and antisense) targeting the promoter locus of Foxm1 (5′-GCCGATTGGCGACGCT-3′ and 5′-CGCCGCTTTCAGTTGTTCCG-3′) or Tgfb1 (5′-CCTTGACACTCTCATCCGCA-3′ and 5′-TGGGACTTCGTGAGAAAACAGA-3′). For ChIP assay with the anti-KLF4 antibody, the amount of coprecipitated DNA was normalized to that from ChIP using control rabbit IgG (1:500, Cell Signaling). For ChIP assay with the H3K9Ac antibody, the amount of coprecipitated DNA in PDGFR-β⁺ cells is relative to the amount of PCR product amplified from input samples reserved before immunoprecipitation and then normalized to this value in SMCs.

Transfected GCs were lysed in Laemmli buffer containing β-mercaptoethanol (BioRad, Hercules, CA, USA). Samples were resolved by 8% Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis, transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Arlington Heights, IL, USA), and immunoblotted with anti-KLF4 antibody (4038, Cell Signaling Tech., Inc., Danvers, MA, USA) diluted 1:200. The membranes were washed and blotted with peroxidase-conjugated donkey anti-rabbit secondary antibody (1:2000) (Boehringer Mannheim, Indianapolis, IN, USA). Immunolabeled proteins were detected using an enhanced chemiluminescence kit (Pierce Chemical Co., Rockford, IL, USA). The 65-kDa KLF4 protein is indicated in Supplementary Figure S1D. To ensure that the lysates were loaded equally, the blots were stripped and incubated with an anti-β-actin antibody (1:4000) (Sigma, St. Louis, MO, USA).