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. Author manuscript; available in PMC: 2015 Sep 4.
Published in final edited form as: Nat Commun. 2015 Mar 4;6:6428. doi: 10.1038/ncomms7428

Fig. 6. Methylation of Arginine 508 is required for SAP145-SMN interaction.

Fig. 6

(a) SMN interacts with SAP145 in vivo. Endogenous Co-IP was performed by immunoprecipitating HeLa cell nuclear extracts using αSMN antibody, followed by western blotting detection using αSAP145 antibody.

(b) Tudor domain of SMN interacts with wild type but not R508K mutant form of SAP145. HeLa cell were transfected with either GFP-SAP145 or GFP-SAP145 (R508K). GST pull-down experiment was performed by incubating transfected cell lysates with recombinant GST-Tudor domains of SPF30, SMN and TDRD3. Eluted samples were detected by western blotting with αGFP antibody. Ponceau S staining displays the loading of total cell lysates and recombinant proteins

(c) SMN interacts with the wild type but not the R508K mutant form of SAP145. HeLa cells were transfected with either GFP-SAP145 or GFP-SAP145 (R508K). The nuclear extracts were immunoprecipitated with αSMN antibody. The eluted samples and input samples were detected with the indicated antibodies.

(d) Overexpression of PRMT9 enhances the interaction of SMN with GFP-SAP145. HeLa cells were transfected with the indicated plasmids. The nuclear extracts were immunoprecipitated with αSMN antibody. The eluted samples and input samples were detected with the indicated antibodies.

(e) An intact SAP145 R508 methylation site is required for U2 snRNP maturation. HeLa cells were transfected with either GFP-SAP145 or GFP-SAP145 (R508K). Cell lysates were immunoprecipitated with αGFP antibody. The eluted samples and input samples were detected with αSmB (Y12) and with αGFP antibodies.