Fig 1.

PHF8 protein levels are regulated by the ubiquitin-proteasome system, with the highest levels being found at G₂ and M phases. (A and B) HeLa cells were synchronized at M phase and harvested at 2-h intervals upon release for 24 h. (A) Total cell lysates were probed for PHF8, the G₂/M marker cyclin B1, and the G₁/S marker cyclin E1. (B) ImageJ quantification of data in panel A was carried out with normalization against actin, and the highest protein level was set to 1. thy-noc, thymidine-nocodazole. (C) Flag-HA-PHF8 was transfected with the empty vector or HIS-Ub and treated with DMSO or MG132. Denatured lysates underwent HA IP and were probed for HIS-Ub. The extent of ubiquitylation in each lane was quantified by ImageJ and normalized against the input, and the quantification is presented below the HIS-Ub blot. WB, Western blotting. (D) Endogenous PHF8 half-life was assessed by cycloheximide (CHX) chase. A total of 100 μg/ml of cycloheximide and 10 μM MG132 or an equal volume of DMSO were added, and cells were harvested at 0, 2, 4, or 8 h. ImageJ was used to quantify the amount of PHF8 (normalized against actin), which is presented below the PHF8 blot.