Psip1/Ledgf p52 Binds Methylated Histone H3K36 and Splicing Factors and Contributes to the Regulation of Alternative Splicing (original) (raw)
Figure 3
Immunoprecipitation of Psip1/p52 and p75.
A) Immunoblot of NIH 3T3 nuclear extract with antibodies; A300-848 which recognizes only the p75 isoform of Psip1, and A300-847 which detects both p52 and p75. B) IPs with IgG, A300-847 and A300-848 from NIH 3T3 nuclear extracts, immunoblotted with antibodies recognizing p75 (A300-848) or p52 (A300-847). Input is 5% total extract. C) Immunoblot of A300-847 IPs with αSRSF1. IP was also performed in the presence of RNase A. Input is 10% of total extract and IgG served as a control. D) In vitro pulldown of 293T cell expressed T7-SRSF1 using GST-p52 and Psip1-PWWP and immunoblotted with αT7. Input is 5% of T7-SRSF1 and GST alone is control. E) In vitro pulldown with GST-p52 of; T7-SRSF1 and mutants that mimic its hypo-(RG) and hyper-phosporylation (RD), T7-SRSF3 and GFP-SRSF2. Immunoblotting was with αT7 or αGFP. F) ChIP with αH3K36me3 from wild-type (wt) and _Psip1_−/− MEFs immunoblotted with antibodies detecting Srsf1, Srsf2, Srsf3, PTB, Psip1 and H3K36me3. G) In vitro pulldown of HeLa core histones by T7-SRSF1 in the presence or absence of Psip1/p52 and immunoblotted with antibodies detecting pan H3, H3K36me3, H3K9me2 and H3K4me3.