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Fig. 5 Coimmunoprecipitation (Co-IP) assay of Hsp70 binding with myosin chaperone Unc45b. (A) HEK293 cells were transfected with DNA plasmid expressing Myc-tagged Hsp70.3 or Hspa8 alone or co-transfected with plasmid expressing FLAG-tagged myosin chaperone UNC45b. Protein extract of the transfected cells was used for immunoprecipitation with anti-FLAG antibody and western analysis using anti-Myc and anti-FLAG antibodies. Co-IP results showed that Hsp70.3 and Hspa8 were coprecipitated with Unc45b. (B) To assess whether this coprecipitation correlated with the unregulated Hsp70s, the Co-IP assay was performed with two upregulated cytosolic Hsp70s (Hspa1b and Hsc70), and the mitochondria Hspa9 which was not upregulated in smyd1b mutant embryos. The Co-IP results showed that Hspa1b and Hsc70 were coprecipitated with Unc45b. In contrast, the mitochondrial Hspa9 that was not coprecipitated with Unc45b.