Figure 3

Western blotting and chromatin immunoprecipitation (ChIP) studies. (A): PTPRR-1 protein expression in total cell extracts from the cell lines used for ChIP studies (Colo205 and SK-N-SH) are shown. Proteins were separated on a 10% polyacrylamide gel. Reactivation of PTPRR protein expression was also investigated in Colo205 cells after treatment with 5-Aza-2'-deoxycytidine (5-Aza-dC) or 3-Deazaneplanocin A (DZNep) plus Trichostatin A (TSA). Extracts of 293T cells transiently transfected with PTPRR-1 cDNA were used as positive controls. PTPRR expression was also documented in assays of protein extracts from epithelial cells of normal colonic crypts, as expected. (The slightly higher molecular weight might be due to a post-translational modification occurring in vivo in this tissue.) MSH6 antibodies were used as proliferation-rate controls: In cells treated with drugs, the expression of the DNA mismatch repair protein MSH6 was decreased --- as expected --- due to reduced cell viability. (B): Immunoprecipitated DNA was quantified by real-time PCR. Enrichment was calculated as percentage of input DNA control (details in Methods). Upper 3 panels: Chromatin prepared from Colo205 cells was immunoprecipitated with antibodies against H3K9ac (acetylated-histone H3Lys9, white bars), H3K27me3 (trimethyl-histone H3Lys27, light gray bars), and H3K9me3 (trimethyl-histone H3Lys9, dark gray bars). The specifity of immunoprecipitations was verified by the selective enrichment, in the PCR amplifications, of the actively transcribed RPL30 gene (H3K9ac antibody), the polycomb target KCNA1 promoter (H3K27me3 antibody), and the heterochromatic Sat 2 locus (H3K9me3 antibody). Lower panel: PTPRR-1 promoter histone code differences between the colorectal cancer cell line Colo205 and the neuroblastoma cell line SK-N-SH. (PTPRR-1 transcript and PTPRR-1 protein expression profiles for the 2 lines are shown in Figures 2B and 3A, respectively.)