Anti klf4

Subcellular fractions were prepared with the ProteoExtract subcellular proteome extraction kit (Calbiochem) following manufacturer’s instructions. Nuclear and total soluble cell-fraction lysates were quantified using the Pierce BCA protein assay (Thermo Scientific). Proteins were separated by SDS-PAGE on a NuPAGE mini gel (Invitrogen). Proteins were transferred to a PVDF membrane, blocked with 5% nonfat dry milk or Bovine Serum Albumin (Sigma) in TBST for 1 hr RT, primary antibody was added and incubated O/N at 4 °C. Then blot was washed 3X in TBST, then incubated in 5% nonfat dry milk in TBST with secondary-HRP conjugated antibody for 1 hr RT, washed 3X in TBST, developed with SuperSignal West Pico chemiluminescence kit (Thermo Scientific) and exposed to Biomax MR film (Kodak). Western blots were performed with the following antibodies: anti-beta actin (Sigma), anti-EGR1 and anti-Hes1 (Santa Cruz), anti-GFAP and anti-Histone H3, anti-KLF4, anti-SOX2 (R&D), anti-nestin (Covance), anti-beta-tubulin (Sigma).

LNCaP and DU145 cells (1 × 10⁷ cells) were fixed with 1% formaldehyde for 10 min before quenching with glycine (0.125 M) for 5 min. The nuclei were isolated using nuclear isolation buffer (150 mM NaCl, 10 mM HEPES pH 7.5, 1.5 mM MgCl₂, 10 mM KCl, 0.5% Nonidet P-40 and 0.5 mM dithiothreitol) and resuspended in nuclear lysis buffer (50 mM Tris-Cl pH 8.1, 10 mM EDTA and 1% SDS). Nucleic lysates were sonicated in order to get DNA fragments ranging from 200 bp to 600 bp. The input DNA controls (10%) were frozen after sonication until further processing. After blocking of unspecific DNA with salmon sperm DNA for 1 hour at 4 °C, the sonicated nuclear material was immunoprecipitated using specific histone antibodies (anti-H3K27me3, −H3K9me3, and –H3K4me3 from Abcam, and anti-KLF4 from R&D Systems) by adding the protein A-conjugated Sepharose (Amersham Biosciences and GE Biosciences). One sample per cell line was incubated without antibodies and used as negative control. After immunoprecipitation over night at 4 °C, protein-DNA complexes bound to Sepharose were washed, and the DNA was isolated according to standard ChIP procedure (Abcam). Enrichment of specific post-translational histone modifications and of KLF4 in IGF2-DMR0 was analyzed by qPCR with IGF2-DMR0 specific primers (Table 2). Input DNA was used as calibrator.