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. 2008 Apr 16;82(13):6734–6746. doi: 10.1128/JVI.00342-08

FIG. 1.

FIG. 1.

The carboxy terminus of LANA binds with NuMA. (A) Schematic showing the domains of the LANA protein. LANA contains two nuclear localization sequences (NLS), a proline-rich domain (P-rich) and a glutamine-rich repeat region (Q-rich), an acidic domain (AD), and a putative leucine zipper (L-Zipper). C-terminal LANA mediates TR DNA binding, Brd2, MeCP2, pRb binding, and chromosome association. Brd2, bromodomain containing 2. (B) GST, GST-LANA-N, and GST-LANA-C fusion proteins were expressed in Escherichia coli and purified with glutathione-Sepharose beads. Nuclear extracts from BCBL-1 cells were incubated with either GST control or GST-LANA truncations normalized by Coomassie staining. The precipitated proteins were resolved by SDS-PAGE and detected with goat anti-NuMA antibody. In each case, 5% of the cell lysates were used as input for comparison. PC, precleared fraction. (C) Twenty million HEK293T cells were transfected with 20 μg of GFP-NuMA and 20 μg of either pA3M-LANA-C or pA3M-LANA-N expression plasmid. The cells were harvested at 36 h posttransfection, and the lysates were immunoprecipitated (IP) with 1 μg of anti-Myc antibody. Samples were resolved on SDS-6% PAGE and probed with anti-NuMA antibody. LANA truncations were detected with 9E10 Myc hybridoma supernatant. (D) Twenty million HEK293T cells were transfected with 20 μg of GFP-NuMA and 20 μg of pA3M-LANA expression plasmids as indicated. The cells were harvested at 36 h and were immunoprecipitated with 1 μg of anti-Myc antibody. Samples were resolved on SDS-6% PAGE and immunoblotted with anti-NuMA antibody to detect NuMA and 9E10 Myc hybridoma for LANA. (E) Fifty million KSHV-positive and negative BCBL1 and BJAB cells, respectively, were harvested and immunoprecipitated with 3 μl of polyclonal anti-NuMA antibody. Reverse immunoprecipitations were performed with 3 μl of rabbit anti-LANA antibody. LANA and NuMA were detected using the respective antibodies. (F) Twenty million HEK293T cells were transfected with 20 μg of GFP-NuMA and 20 μg of either pA3M-LANA aa 840 to 963 or pA3M-LANA aa 840 to 1067 expression plasmid. The cells were harvested at 36 h and immunoprecipitated with 1 μg of anti-Myc antibody.