Figure 6.
(D) Cells were transfected and stimulated as in (A) and cellular protein was isolated from the same TRIzol® reagent preparations used for RNA extraction. Protein was separated by 15% SDS–PAGE and transferred to PVDF membrane for immunoblotting with an H3K36me3 (i)-specific antibody. Ponceau S staining of histones was performed to verify even loading (ii). (E) C3H 10T½ cells were untransfected (−) or transfected with Setd2, non-targeting (non-t) or NSD1 siRNA. Cells were quiesced 24 h later, and after a further 24 h left untreated (con) or treated with EGF (50 ng/ml) for 15–60 min. Total mRNA was isolated and northern blotting carried out with 3′ c-fos (i) and 3′ c-jun (ii) probes and a full-length gapdh cDNA probe (iii).