FIGURE 2.
MRI-2 interacts with Ku. A, MRI-FLAG was co-immunoprecipitated (IP) from HEK 293T cells and analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. The co-IP of MRI-2 (†) enriched two bands (*), which were identified as Ku70 (XRCC6) and Ku80 (XRCC5) by mass spectrometry (data not shown). MW, molecular weight markers. B, semiquantitative proteomics by spectral counting was used to confirm MRI-2 interaction partners. Bands of interest were excised from experimental and control samples by cutting straight across the gel and subjected to in-gel digest and proteomics. The top 10 most abundant proteins in each experimental sample were plotted in terms of spectral counts and compared with the negative control. Ku70 and Ku80 were enriched 3–10-fold. C, confirmation of MRI-2 interaction with Ku70 and Ku80 by Western blotting. FLAG-tagged MRI-2 or empty vector was immunoprecipitated from HEK 293T cells and then analyzed by Western blotting with antibodies against Ku70 and Ku80. Lysates were simultaneously probed to confirm equal loading. Enrichment of Ku70 and Ku80 by the MRI-2 co-IP as compared with vector background was observed. D, co-immunoprecipitation of MRI-1, MRI-2, and MRI-3 (indicated by arrowhead on gel) from HEK293T, with SDS-PAGE analysis and Coomassie Brilliant Blue staining. The four molecular mass regions analyzed were 70, 80, and 450 kDa (bands of interest indicated by *). E and F, semiquantitative proteomics by spectral counting demonstrates enrichment of Ku70 and Ku80 by MRI-1 (E) but not MRI-3 (F). G, spectral counting for the 450-kDa band for all three proteins shows enrichment of DNA-PK by MRI-1. H, co-IP and Western blotting confirm interactions of MRI-1 with Ku and DNA-PK and the absence of interactions with MRI-3. Lysates were analyzed to demonstrate equal loading.