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. 2016 Mar 3;61(5):734–746. doi: 10.1016/j.molcel.2016.02.008

Figure 5.

Figure 5

RNMT Phosphorylation Reduces Inhibition by KPNA2

(A) HeLa cells expressing HA-RNMT WT, T77A, T77D, or vector control (C) were transfected with pcDNA5 Myc-KPNA2 or vector control (C). 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WBs were performed to detect HA-RNMT, RNMT, and Myc-KPNA2.

(B) KPNA2 IP was performed on HeLa cells expressing HA-RNMT WT, T77A, T77D, or a vector control using anti-KPNA2 antibody, and KPNA2 and RNMT were detected by WB.

(C) HeLa cells were transfected with pcDNA5 Myc-KPNA2. Cells were released from double thymidine block for 2 hr (S) or 8 hr (G2/M), including incubation with 9 μM RO-3306 for 15 min (G2/M RO). Asynchronous cells transfected with the vector control were used as a control. 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WBs were performed to detect RNMT and KPNA2.

(D) HeLa cells were treated as in (C), except that G2/M cells were also treated with 50 μM roscovitine (G2/M Rosc) for 15 min. Anti-KPNA2 antibodies were used to immunoprecipitate KPNA2 from cell extracts using anti-Tubulin antibodies as a control. WBs were performed to detect KPNA2, RNMT, and Tubulin in extracts and immunoprecipitates.

(E) A cap methyltransferase assay was performed using 40 nM recombinant RNMT and titration of recombinant KPNA2 or BSA. Activity is reported relative to the RNMT control.

(F) Recombinant RNMT and RAM were incubated with recombinant GST-KPNA2 WT, 1–455, 72–529, or GST alone, and complexes were affinity-purified with glutathione-Sepharose. Inputs and eluates were resolved by SDS-PAGE and Coomassie blue-stained, and RAM was detected by WB.

(G) HeLa cells were transfected with pcDNA5 Myc-KPNA2 WT, 1–455, 72–529, or vector control (C). 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WB was performed to detect Myc-KPNA2, KPNA2, RNMT, and Tubulin. (Note that the KPNA2 antibody raised against the N terminus does not recognize KPNA2 72–529.)

(H) A cap methyltransferase assay was performed using recombinant RNMT-RAM and recombinant KPNA2 WT, 1–455, 72–529, or GST alone. The charts depict the average and SD of four experiments. ∗∗p < 0.005, t test. Nonspecific bands are indicated with a star.

See also Figures S4, S5, S6, and S7.