Figure 1.

BAG3 is phosphorylated at T285 during mitosis. (A) Schematic representation of BAG3 modular domains and phosphorylated residues, as identified by AP-MS. See also Figure S1 for MS data. Sequence alignments show the interspecies conservation of the identified phosphorylated motif. BAG3-GFP^DMB and BAG3-GFP^QMB constructs were generated by alanine substitution of designated residues (box). (B) Western blots of BAG3-GFP IPs prepared from HeLa-RFP-H2B cells growing asynchronously (AS) or arrested in mitosis by nocodazole treatment (M) and recovered by a mitotic shake-off, showing the mitotic mobility shift of BAG3-GFP constructs. Cells were transfected with BAG3-specific siRNA and transduced with Ad-BAG3-GFP. (C) Representative Western blots of HeLa cell extracts that have been transfected with control siRNA or BAG3-specific siRNA targeting BAG3 open reading frame (siBAG3 [ORF]), showing the pT285-BAG3 antibody specificity; M: nocodazole-arrested cells recovered by a mitotic shake-off; AS: asynchronous cells; levels of total BAG3 and GAPDH (loading control) are shown. (D) BAG3-GFP IPs were prepared from asynchronous (AS) or from mitotic HeLa-RFP-H2B cells synchronized by nocodazole and recovered by mitotic shake-off after transduction of cells with Ad-GFP-BAG3, as indicated; Western blots show total levels of GFP-BAG3 proteins and pT285-BAG3. (E) Extracts were prepared from asynchronous (AS) or from mitotic HeLa-RFP-H2B cells synchronized in early mitosis by nocodazole or arrested at the G2/M stage by RO3306. Western blots of endogenous proteins were performed as indicated; GAPDH: loading control. (F) Schematic of the protocol used. HeLa-RFP-H2B cells synchronized in mitosis by nocodazole, were recovered by a mitotic shake-off, and then seeded on poly-L-lysine-coated dishes in fresh medium, with or without MG132 (5 µM) to inhibit mitotic exit. (G) Cells were collected at different times and extracts were analyzed by Western blots, as indicated; GAPDH: loading control.