FIGURE 4:

The SIM within the N-terminal domain of p150 is required to maintain Ki-67 localization to the PCL. (A) HeLa cells in prometaphase expressing the indicated shRNA-resistant p150 transgene were infected with lentiviruses encoding the indicated shRNAs (sh-Luc or sh-p150) for 72 h and then stained with DAPI to visualize DNA (blue) and antibodies against Ki-67 (green). Note that upon expression of sh-p150, cells expressing luciferase (V5-Luc), the p150 C-terminus (V5-(p150-311-938)), or p150 with a I99A point mutation in the SIM motif required longer exposure times to detect Ki-67 on the PCL. Scale bar, 10 μm. (B) Immunoblots of cell extracts described in A expressing either sh-Luc (L) or ph-p150 (P). Blots were probed with antibodies recognizing p150 (top), V5 transgenes (middle), and fibrillarin (loading control; bottom). Note the depletion of endogenous p150 in sh-p150 lanes and that V5-tagged transgene expression often increased in these lanes, as we had previously (Smith et al., 2014).