Fig. 3

The TPR domain facilitates CLUH self-interaction and its interaction with SPAG5. A Western blot analysis of co-IP, between GFP- and 3xHA-tagged CLUH proteins stably expressed in HCT116 cells. The IP is performed using magnetic beads coupled with anti-HA antibodies (IP-HA) with protein extracts treated (+) or not (−) with RNaseA/T1. The proteins are detected using anti-HA and anti-GFP antibodies. GAPDH and ACTIN are used as specificity controls. The loaded samples correspond to 0.5% of the input and 20% of the pulled-down samples. B Ethidium bromide-stained agarose gel loaded with RNase treated (+) or non-treated (−) total protein extracts used for the IP showing the presence of ribosomal RNA. C Scheme showing the human CLUH protein domains identified using Pfam database. The amino acid positions of each domain are indicated. The mutant protein CLUH∆TPR has been generated by deleting the TPR domains. D Western blot analysis of co-IP, between 3xHA-tagged wildtype CLUH (in orange) or the CLUH∆TPR mutant (in red) with the endogenous CLUH and SPAG5 proteins. A 30-kDa tag corresponding to the BioID2 protein followed by 3xHA peptide is added in N-terminal (Tag-3xHA) of each transgene. A GFP tagged construct is used as a specificity control. The co-IP is performed on total extracts (INPUT) from HCT116 cells stably expressing the different constructs using magnetic beads coupled with anti-HA antibodies (IP-HA). The size of the endogenous CLUH (black), wildtype transgene (orange), and the delta-TPR mutant (red) is indicated. The indicated proteins are revealed using specific antibodies. TUBULIN Is used as a loading control. The loaded samples correspond to 0.5% of the input and 20% of the pulled-down samples