HJURP is required for CENP-A localization to centromeres. (A) The centromeric association of CENP-A is lost in cells depleted of HJURP. HeLa cells were transfected with either scrambled siRNA (SsiRNA) or with CENP-A siRNA or with HJURP siRNA1 and siRNA2. Seventy-two hr posttransfection cells were immunostained (Green) with anti-CENP-A antibody and DAPI staining (Blue). (B) Western blot analysis of the depletion of HJURP and CENP-A upon treatment with siRNA. HeLa cells were transfected with the respective siRNA and 72 hr posttransfection they were harvested, total cell extracts were prepared, and the presence of HJURP and CENP-A was detected by Western blotting using antiHJURP and anti-CENP-A antibodies. The blot was also revealed with an antiactin antibody as a control for equal loading. (Left) Depletion of both CENP-A and HJURP upon treatment with siRNA against HJURP. (Right) Depletion of CENP-A upon treatment with siRNA against CENP-A. Ctrl, nonsiRNA treated cells; Ssi, cells treated with scrumble siRNA; Si1 and Si2, cells treated with two distinct (Si1 or Si2) siRNAs against HJURP (Si1 and Si2 siRNA were used to suppress the expression of HJURP in the experiments presented in (A).