Journal: Journal of Biological Chemistry

Article Title: Identification of the Drosophila Ortholog of HSPB8

doi: 10.1074/jbc.m110.127498

Figure Lengend Snippet: FIGURE 1. D. melanogaster HSP67Bc is a functional ortholog of human HSPB8. A and B, human HSPB8 and Dm-HSP67Bc co-immunoprecipitate with Dm-Starvin. HEK-293T cells were transfected with vectors encoding for D. melanogaster Myc-Starvin alone or together with either V5-HSPB8, V5-HSP67Bc, V5-L(2)efl, or V5-CG14207. 24 h post-transfection, the cell lysates were subjected to immunoprecipitation (IP) with an antibody against the V5 tag, and the immunoprecipitated complexes were analyzed by Western blotting (WB) using V5- and Myc-specific antibodies. Among the D. melanogaster sHSPs analyzed, HSP67Bc interacts with Dm-Starvin (B), similarly to human HSPB8 (A). C, like HSPB8, Dm-HSP67Bc also binds to BAG3, the human functional ortholog of Dm-Starvin. HEK293 cells were transfected with vectors encoding for human Myc-BAG3 alone or together with either V5-HSP67Bc, V5-L(2)efl, V5-CG14207, or V5-HSPB8 and V5-HSP70, both used as positive controls and subjected, 24 h post-transfection, to immu- noprecipitation with a V5-specific antibody. D, endogenous Hsp67Bc interacts with Starvin in vivo in fly head extracts. V5-starvin was expressed in flies under the control of the grm-GAL4 driver. Immunopre- cipitation with a specific V5 antibody was carried out using fly head protein extracts from control flies (grm/) and flies expressing V5-Starvin (gmr/V5-Stv). Interaction of endogenous Hsp67Bc with V5-Starvin was investigated by Western blotting using a specific rabbit polyclonal Hsp67Bc antibody. E, total levels of HSP67Bc are increased when it is co-expressed with Starvin. Drosophila Schneider S2 cells were trans- fected with vectors encoding for V5-HSP67Bc, V5-L(2)efl, and V5-CG14207 alone or in combination with V5-Starvin. The protein expression levels were analyzed by Western blotting 48 h post-transfection (aver- age values S.E. (error bars) of n 3–4 independent samples). F, human muscle tissue section showing that endogenous HSPB8 colocalizes with -actinin at the Z band. G, endogenous Dm-HSP67Bc colocalizes with -actinin at the Z band in third instar larvae muscles.

Article Snippet: Mouse monoclonal anti- -actinin and mouse monoclonal anti- -tubulin were from Sigma-Aldrich, whereas mouse monoclonal anti-Myc (9E10)was from theAmericanTypeCulture Collection.Mousemonoclonal anti-total-eIF2 and rabbit polyclonal anti-phospho-eIF2 were from Cell Signaling and Sigma-Aldrich, respectively.

Techniques: Functional Assay, Transfection, Immunoprecipitation, Western Blot, In Vivo, Control, Expressing, Muscles