Skip to main content
. 2015 Oct 23;11(10):e1005582. doi: 10.1371/journal.pgen.1005582

Fig 6. Depletion of p62, which forms a mitotic complex with BAG3-HSPB8, recapitulates the same phenotype on spindle dynamics.

Fig 6

(A) Representative Western blots of total extracts prepared from HeLa-RFP-H2B cells transfected with control siRNA (siCtl) or p62-specific siRNAs (sip62 #1, #2); vinculin and GAPDH levels: loading controls. (B) Confocal time-lapse sequences from HeLa-RFP-H2B cells transfected with sip62_1 and transduced with BacMam-GFP-a-tubulin and BacMam-RFP-actin, which show aberrant spindle rocking; colored asterisks designate spindle poles. Cells were synchronized by a double Thymidine block and imaged by spinning disk confocal microscopy for 60 min at ~1.5 min intervals; Bars, 10 μm. (C) Quantification of cells from B, indicating abnormal mitosis defined as spindle rocking, stalled in mitosis +/- spindle rocking, or others (chromosome misalignment or multipolar spindle); means +/- SE from at least 215 cells from at least 3 independent experiments, as indicated. (D, E) BAG3 IPs were prepared from asynchronous (AS) or from mitotic HeLa-RFP-H2B cells synchronized by a Thymidine/nocodazole block and recovered by mitotic shake off (M), or from cells treated with proteasome inhibitor for 16 h (MG132, 5 μM), using anti-BAG3 antibody or rabbit IgG, and analyzed by Western blot with the indicated antibodies. The levels of BAG3, HSPB8, p62, HDAC6, HSP70 and HSC70 in total cell extracts are shown (Input); phospho-histone H3 (pH3) and histone H3 levels: mitotic markers. The data are representative of at least 3 independent experiments.