Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency
Figure 3
Acetylation regulates NPM interaction with LANA.
(A) Whole cell extracts of BC-3 and BCBL-1 cells stably expressing sh-Scr, or two different constructs of sh-RNA specific for NPM (sh1-NPM and sh2-NPM) were analyzed by immunoblotting with anti-NPM antibodies. Fibrillarin and tubulin were used as loading controls. (B) Extracts of BCBL-1 cells untreated (ctrl), NaB treated (NaB), or cells stably expressing sh1-NPM or sh2-NPM were separated by SDS-PAGE followed by immunoblotting with antibodies against LANA, HDAC1 and tubulin. These samples represent the input (10%) for panels D and E. (C) Whole-cell extracts of ctrl and NaB-treated BCBL-1 cells from B were reciprocally immunoprecipitated using anti-HDAC1 or anti-NPM antibodies. Rabbit or mouse normal IgG (IgG) served as a control for immunoprecipitation. Protein complexes were resolved by SDS-PAGE and followed by immunoblotting with anti-NPM and anti-HDAC1 antibodies. Input (10%) is shown on the right. (D) Whole cell extracts of BCBL-1 cells in B were immunoprecipitated with anti-HDAC1 antibodies. Normal rabbit IgG was used as a negative control. The immunocomplexes were separated by SDS-PAGE followed by immunoblotting with antibodies against LANA and HDAC1. (E) Whole cell extracts of BCBL-1 cells used in panel B were immunoprecipitated with anti-acetyl lysine antibody and analyzed for indicated proteins by immunoblotting. Rabbit IgG served as a negative control for immunoprecipitation. (F) BC-3 cells were treated with vehicle (ctrl) or 1 mM sodium butyrate (NaB) for 24 hours. Whole-cell extracts were subjected to immunoprecipitation using anti-NPM (NPM) or normal mouse control IgG (IgG). The samples were separated by SDS-PAGE and followed by immunoblotting with anti-acetyl-K lysine and anti-NPM antibodies. (G) Whole-cell extracts of ctrl and NaB-treated (24 hours) BCBL-1 cells from B were subjected to immunoprecipitation using anti-NPM (NPM) or normal mouse control IgG (IgG). The samples were separated by SDS-PAGE and followed by immunoblotting with anti-p300 and anti-NPM antibodies. (H) Whole cell extracts of BCBL-1 cells untreated (ctrl), or NaB-treated (NaB), were immunoprecipitated with anti-LANA antibodies (left panel). Protein complexes were resolved by SDS-PAGE and followed by immunoblotting with anti-NPM and -LANA antibodies. Input (10%) is indicated in the right panel. Arrowheads indicate the position of possibly acetylated LANA. For the purpose of clarity of the data Western blots lanes (derived from the same SDS-PAGE gel) were reorganized as indicated by black dashed bars.