FIG. 5.
ChIP. (A) ChIP and Western blotting (WB) with BC-3 cells. In vivo cross-linked chromatin was digested with micrococcal nuclease (DNA length of < 1kb) and immunoprecipitated with 1 μg of a rat anti-LANA antibody or control rat immunoglobulin (Dako). The precipitated matrix was analyzed by Western blotting with an anti-LANA antibody, an anti-HP-1α antibody (Euromedex), and an anti-HP-1β antibody (Euromedex). IgL, light chain of immunoglobulin. (B) Chromatin states of the TR and the K1 and LANA promoters. PCR was performed with primers for the TR, the K1 gene, and the promoter of the latent transcript (LANA) after ChIP. The amplified products were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide (Inp, 5% of input; C, control rat immunoglobulin; LN, rat anti-LANA monoclonal antibody [Advanced Biotechnologies Inc.]; HP, mouse anti-HP-1α monoclonal antibody [Upstate]; H3MeK9, mouse anti-methylated histone H3 monoclonal antibody [Upstate]). (C) The KSHV ORF50/RTA region was specifically precipitated by the anti-HP-1α antibody in the ChIP assay. Precipitated DNA was reacted in the PCR with the primers for the ORF50/RTA locus (panels 1 and 2) or the ORF73/LANA locus (panels 3 and 4), and the results of 2% agarose-TBE electrophoresis are shown. A schematic presentation of KSHV ORF50/RTA and ORF73/LANA is also shown. Arrows indicated the priming site of each PCR primer.