Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2013 Sep 28.

Published in final edited form as: Oncogene. 2012 Jun 4;32(13):1691–1701. doi: 10.1038/onc.2012.182

(A) Stability of p27 in DAOY, D283, and UW228 cells was measured following treatment with cycloheximide (CHX) (100 µg/ml) for various time periods (0–120 mins) or MG132 (20 µM) for 4 hrs and analyzed by Western blotting. GAPDH was used as a loading control. (B) REST-dependent changes in p27 ubiquitination were assessed by immunoprecipitation experiments following transient transfection of DAOY cells with REST-specific siRNA or scr siRNA. p27 was immunoprecipitated from whole cell extracts using anti-ubiquitin (Ub) antibody followed by Western blotting using anti-p27 antibody. Input lanes (0.5% and 10%) are included. (C) Change in USP37 gene expression in DAOY and D283 cells transfected with REST-specific siRNA or scr siRNA was determined by SYBR-Green Q-RT-PCR analysis using specific primers. Changes in REST and USP37 gene expression were plotted relative to 18S RNA. Each experiment was performed in triplicate and the standard error calculated. Significance (* p<0.1, ** p<0.05, *** p< 0.001) was calculated using Statistica 6.0 software. (D) USP37 expression in normal cerebella and established or primary medulloblastoma cultures were measured by Q-RT-PCR analyses and normalized to 18S levels. (E) REST binding to the RE1 element downstream of the USP37 gene was evaluated by chromatin immunoprecipitation assay. REST was immunoprecipitated from formaldehyde-cross-linked and sonicated nuclear DAOY cell extracts, and the associated DNA was measured by Q-RT-PCR analysis. The results shown are the average of three separate experiments. Significance (* p<0.1, ** p<0.05, *** p< 0.001) was calculated using Statistica 6.0 software.

(A) Stability of p27 in DAOY, D283, and UW228 cells was measured following treatment with cycloheximide (CHX) (100 µg/ml) for various time periods (0–120 mins) or MG132 (20 µM) for 4 hrs and analyzed by Western blotting. GAPDH was used as a loading control. (B) REST-dependent changes in p27 ubiquitination were assessed by immunoprecipitation experiments following transient transfection of DAOY cells with REST-specific siRNA or scr siRNA. p27 was immunoprecipitated from whole cell extracts using anti-ubiquitin (Ub) antibody followed by Western blotting using anti-p27 antibody. Input lanes (0.5% and 10%) are included. (C) Change in USP37 gene expression in DAOY and D283 cells transfected with REST-specific siRNA or scr siRNA was determined by SYBR-Green Q-RT-PCR analysis using specific primers. Changes in REST and USP37 gene expression were plotted relative to 18S RNA. Each experiment was performed in triplicate and the standard error calculated. Significance (* p<0.1, ** p<0.05, *** p< 0.001) was calculated using Statistica 6.0 software. (D) USP37 expression in normal cerebella and established or primary medulloblastoma cultures were measured by Q-RT-PCR analyses and normalized to 18S levels. (E) REST binding to the RE1 element downstream of the USP37 gene was evaluated by chromatin immunoprecipitation assay. REST was immunoprecipitated from formaldehyde-cross-linked and sonicated nuclear DAOY cell extracts, and the associated DNA was measured by Q-RT-PCR analysis. The results shown are the average of three separate experiments. Significance (* p<0.1, ** p<0.05, *** p< 0.001) was calculated using Statistica 6.0 software.