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. 2010 Mar 19;6(3):e1000818. doi: 10.1371/journal.ppat.1000818

Figure 2. NPM interaction with LANA is dependent on Thr199 phosphorylation of NPM by v-cyclin-CDK6.

Figure 2

(A) U2OS cells stably expressing GFP or LANA were transfected with an expression vector for Myc-v-cyclin or with an empty vector as indicated. Cell extracts (10% of immunoprecipitates) were resolved by SDS-PAGE (left panel) or were immunoprecipitated with anti-LANA (middle panel) or anti-NPM antibodies (right panel). IgG immunoprecipitations were included as controls. Immunoblotting was performed with anti-NPM and –LANA as indicated. Asterisks indicate the nonspecific crossreactivity detected with NPM and LANA antibodies. (B) Gel filtration fractions marked 1-3 (ca. 700-440 kDa) of BC-3 cells expressing sh-Scr or sh-CDK6 (as described in Figure 1C) were immunoprecipitated with anti-LANA antibodies, separated by SDS-PAGE and followed by immunoblotting with antibodies against NPM and LANA. Input (10%) is indicated on the right. (C) U2OS cells stably expressing LANA were transfected with the Myc-v-cyclin or empty vector, and the NPM expression constructs for wt (eGFP-NPM) and phosphorylation site mutants eGFP-NPM T4A and T3A as shown on top. Cell extracts (10% of immunoprecipitates) were analyzed by SDS-PAGE (left panel) for the input or were immunoprecipitated with antibodies against LANA or control rat IgG (right panel). The immunoblots were probed with with anti-GFP and –LANA as indicated.