Fig. 3

Validation of NOA36 and HSPs interaction by co-immunoprecipitation. (A) Scheme of the domains of the HSP70 family of proteins. Three domains have been characterized in these proteins: The Nucleotide Binding Domain (NBD), the Substrate Binding Domain (SBD) and the Lid Domain (LD) (B) Western blot analysis of input (left blots) and pull-downs with anti-FLAG magnetic beads (right blots) in HEK293 proteins extracts of cells co-transfected with FLAG-NOA36 and HA tagged heat shock proteins HSP90AB, HSPA1 and HSPA8 (lines 4, 5 and 6). The FLAG empty vector was also co-transfected with HA-HSPA8 as a control (line 7). FLAG-NOA36 co-immunoprecipitates with HA-HSPA1 and HSPA8 but there was not a clear interaction with HSP90AB (upper right blot). Note that the FLAG-NOA36 transfection in HEK293 lead to the expected 36 kDa protein but a 40 kDa band is also detected by the anti-FLAG antibody (bottom left blot). Co-expression of truncated HSPA8 containing the NBD domain (D1, line 1 in the blots), the NBD and SBD (D1 + D2, line 2), and the SBD and LD domains (D2 + D3, line 3). FLAG-NOA36 only clearly interacts with the SBD + LD truncated protein. (C) Indirect immunofluorescence of a co-transfection with the constructs FLAG-NOA36 (in red) and HA-HSPA8 (in green) in heat shocked HeLa cells. In blue, DAPI staining showing the nucleoli in the transfected cells (arrow heads). Both recombinant proteins co-localize in the nucleoli of a co-transfected cell (yellow staining in the merged image). Bar, 10 μm