Fig. 3

Evidence that SMCR8 protein is poly-ubiquitinated. a The FL-SMCR8 construct was transfected in 293T cells and immunoprecipitated with α-FLAG antibody-bound agarose. A Western blot of whole cell lysates probed with α-FLAG antibody shows expression of full-length FL-SMCR8 protein plus HMW products consistent with PTMs (left). Probing with α-UBB antibody marks HMW products in immunoprecipitates consistent with either poly-ubiquitinated FL-SMCR8 protein or the presence of other HMW ubiquitinated proteins that co-IP with the SMCR8 complex (right). IP reactions were in the presence or absence of 50 μg/ml RNases. b C-terminal V5-tagged SMCR8 and empty vector or HA-tagged ubiquitin were coexpressed in 293T cells and treated or not treated with the proteasome inhibitor MG132. Expression of SMCR8-V5 protein and empty vector, in the presence but not absence of MG132, produces HMW bands on Western blots that are consistent with post-translational modification of SMCR8 at multiple sites. SMCR8-V5 protein coexpressed with HA-UBB and without MG132 shows the same HMW bands, which increase in signal intensity upon incubation with MG132. c V5- or HA-epitope-tagged SMCR8 was coexpressed with empty vector or FLAG-tagged UBB in 293T cells and incubated overnight in the presence or absence of MG132. Cell lysates were subjected to immunoprecipitation with α-FLAG agarose, followed by Western blotting and probing with α-HA (top left panel), α-V5 (top right) or α-FLAG (bottom left) antibodies. A HMW smear seen in immunoprecipitates is consistent with poly-ubiquitination of tagged SMCR8 proteins. In general, overexpression of ubiquitin does not lead to a significant decrease in full-length SMCR8 protein levels