Figure 4.

RNMT T77 Phosphorylation Inhibits Interaction with KPNA2

(A) HeLa cell extracts were incubated with biotinylated-RNMT 73CGKD(T)PSKKR82 peptide, either T77 unphosphorylated (T77) or phosphorylated (pT77). Peptides and associated proteins were affinity-purified, resolved by SDS-PAGE, and Coomassie blue-stained. Proteins in A and B were identified by mass spectrometry. Mascot scores are reported.

(B) HeLa cells were transfected with pcDNA5 Myc-KPNA2 or vector control, and proteins were affinity-purified from cell extracts on RNMT peptides. Myc-KPNA2 was detected by WB performed using 9E10 antibody.

(C) HeLa cell extracts were affinity-purified on RNMT peptides, and endogenous KPNA2 was detected by WB.

(D) HeLa cells were transfected with pcDNA5 Myc-KPNA2. 9E10 antibodies were used to immunoprecipitate Myc-KPNA2 and anti-HA antibodies used as a control. WBs were performed to detect Myc-KPNA2 and RNMT (adjacent panels are components of the same blot).

(E) Anti-KPNA2 antibody was used to immunoprecipitate KPNA2 from cell extracts, with anti-Tubulin antibody used as a control. WBs were performed to detect KPNA2 and RNMT. The star indicates a non-specific band.

(F) Recombinant GST or GST-KPNA2 was incubated with recombinant (untagged) RNMT and affinity-purified on glutathione-Sepharose. Inputs and proteins eluted were resolved by SDS-PAGE and Coomassie blue-stained or analyzed by WB to detect RNMT and KPNA2.

(G) As (F), except GST-RNMT was incubated with purified recombinant KPNA2.

See also Figures S2 and S3.