Fig. 6. Ectopic expression of CENP-A does not alter the level of CENP-B, CENP-C or hSMC1 that is soluble in the cell or associated with chromatin. The levels of various centromere-kinetochore proteins were compared between a stable CHO cell line induced to express HA-tagged CENP-A over 50 passages and the same cell line uninduced over the same number of passages. Protein samples from whole cells (50,000 cell-equivalents per lane) and detergent-extracted nuclear enrichments (100,000 cell-equivalents per lane) were compared by western analysis. Expression of CENP-A-(HA) was determined using anti-HA antibody and SH-CREST antiserum. SH-CREST also recognizes a non-centromeric 20-25 kDa antigen of unknown identity that displays certain biochemical properties similar to CENP-A (Earnshaw and Rothfield, 1985; Valdivia and Brinkley, 1985; Ouspenski and Brinkley, 1993; He and Brinkley, 1996). CENP-C was detected using a specific antibody and with SH-CREST antiserum. hSMC1 was detected using a specific antiserum. CENP-B was detected using SH-CREST. The subcellular localization of CENP-B was unaltered by CENP-A overexpression and serves as a control for the relative amount of protein loaded in each lane.