Fig. 5

DIP2A recruits HDAC2 to repress the transcription of MGMT. a Cell lysis prepared from D54 cells were incubated with anti-IgG or anti-DIP2A antibodies. Immunoprecipitated material was subjected to SDS-PAGE, and western blot analysis was performed with the indicated antibodies. b NE prepared from GBM cells were incubated with anti-IgG or anti-HDAC2 antibodies. Immunoprecipitated material was subjected to SDS-PAGE, and western blot analysis was performed with the indicated antibodies. c Fast protein liquid chromatography separation of 5 mg of total protein from DIP2A expressors of D54 cells was performed by size-exclusion chromatography on a pre-calibrated Superose 6 Increase 10/300. Fractions 18 through 21, corresponding to an approximate molecular mass of 600 through 400 kDa, were probed for DIP2A, HDAC2, and DMAP1 by immunoblotting. d, e Co-IP was performed using NE prepared from GBM cells transfected with DMAP1-specific siRNA (siDMAP1) or NC for 48 h using anti-IgG or anti-DIP2A antibodies. And western blot analysis was performed with the indicated antibodies. f The left panel shows a schematic illustration of DIP2A and its mutants. In the right panel, D54 cells were transfected with flag-DIP2A, flag-A-DIP2A, or flag-B-DIP2A. Forty-eight hours after transfection, cells were subjected to Co-IP analysis using the indicated antibodies for IP and blotting. g D54 cells were transfected with vector control, flag-DIP2A, flag-A-DIP2A, or flag-B-DIP2A plasmid, respectively. The levels of MGMT were detected 48 h later by WB or qRT-PCR, respectively. h Protein extracts from D54 cells were incubated with immobilized flag, flag-DIP2A, flag-A-DIP2A, or flag-B-DIP2A. The input was 10% of the amount of whole-cell extract incubated with the flag fusion proteins. Western blots were probed with the indicated antibodies. i The protein lysates of D54 cells transfected with Fstl1 or empty vector were subjected to Co-IP analysis using the indicated antibodies for IP and blotting. j The protein lysates of 05MG cells transfected with shFstl1–7 or shCtrl were subjected to Co-IP analysis using the indicated antibodies for IP and blotting. k, l D54 cells were separately transfected with shDIP2A, shFstl1–7, Fstl1, DIP2A, or co-transfected with Fstl1 and shDIP2A or DIP2A. ChIP assays were performed using anti-IgG, or anti-HDAC2 antibodies (l), or specific antibodies against H3K9Ac (k). The eluted DNA was subjected to qRT-PCR with the specific primer set for MGMT. NE: nuclear extracts. Student’s t tests and one-way ANOVA were performed. Data are presented as mean ± SEM in three biological replicates (**P < 0.01, ^#P > 0.05)