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. 2021 Oct 2;10(10):2638. doi: 10.3390/cells10102638

Figure 4.

Figure 4

BAG3 phosphorylation at T285 is regulated by p62 during mitosis. (A) Western blots of extracts from asynchronous (AS) or mitotic HeLa cells (M) synchronized in mitosis by nocodazole and collected by mitotic shake-off. Cells were transfected with control siRNA (siCtl) or p62-specific siRNAs (sip62) and levels of pT285-BAG3, BAG3, p62, and GAPDH (loading control) are shown. Graph depicting the fold change in pT285-BAG3 levels, as estimated relative to cells transfected with control siRNA and normalized relative to GAPDH levels; means ± SE from 3 independent experiments. Statistical significance was analyzed by the Student′s t-Test ****: p < 0.0001. See also Figure S4B that depicts the fold change in BAG3 total levels as estimated relative to cells transfected with control siRNA and normalized relative to GAPDH levels. (B) Western blots of cell extracts prepared from asynchronous (AS) or from mitotic HeLa cells (M) synchronized in mitosis by nocodazole and collected by mitotic shake-off; cells were transfected with control siRNA (siCtl) or BAG3-specific siRNA (siBAG3 [ORF]) or HSPB8-specific siRNAs, and protein levels are shown as indicated. Graphs depicting the fold change in total p62 levels (p62) normalized relative to GAPDH levels, and the fold change in pT269-S272-p62 levels (p-p62) normalized relative p62 total levels, as estimated relative to cells transfected with control siRNA. Data are means ± SE from 3 independent experiments. (C) Scheme recapitulating putative functional relationships: CDK1 phosphorylates p62 that in turn enhances CDK1 activity [59]; the increased mitotic association between the BAG3-HSPB8 chaperone complex and p62 may promote BAG3 phosphorylation and stabilize p62 during mitosis.