Fig. 2

Endogenous CLUH interacts with both SPAG5 and KNSTRN and the CLUH-SPAG5 interaction occurs in cytoplasmic granular structures. A Western blot analysis of co-IP between GFP-tagged KNSTRN or SPAG5 proteins and the endogenous CLUH protein and in HCT116 cells. The co-IP is performed using magnetic beads coupled with anti-GFP antibodies (IP-GFP) on total extracts (INPUT) from cells stably expressing the tagged proteins. Wild-type HCT116 and cells expressing BirA*HA-GFP (TAG-GFP) are used as negative controls. The loaded samples correspond to 1% of the input and 20% of the pulled-down samples. The proteins are revealed using GFP-, CLUH-, and TUBULIN-specific antibodies. TUBULIN is used to control nonspecific binding. Non-specific bands are marked by asterisk (*). B Split-GFP analysis of SPAG5 and CLUH interaction. HCT116 stably expressing SPAG5 fused with sfGFP-1-10 together with CLUH or GAPDH fused with mCherry-sfGFP11 are fixed and analyzed by confocal fluorescent microscopy. Reconstituted sfGFP signal is shown in black (upper panel) and in green (lower panels). The mCherry signal is shown in red and nuclei, stained with Hoechst, are in blue. The colocalization of green and red signal is shown in yellow and indicated with arrows. Scale bar is indicated in white. C Confocal fluorescent microscopy images of HCT116 cells stably expressing GFP fused to SPAG5, KNSTRN, or CLUH. GFP signal is shown in green and nuclei, stained with Hoechst, in blue. Scale bar is indicated in white