Fig. 3

Fig. 3

a Schematic representation of the different SMN protein domains and amino acid residues in the portion of the Tudor domain that contains the SUMO-interacting motif (SIM). The residue positions of the SIM are indicated in blue. K119 is the putative sumoylation site based on SUMOplot analysis. E134 is the position of a mutation found in SMA patients (E134K). The changes in SMN protein sequence to generate SMN-2VA and SMN-E134K mutants are reported in red. b The SIM-like motif of SMN is required to directly bind with SUMO in vitro. Equal protein amounts of purified His-SUMO1 or SUMO-2 (Input) were individually incubated with wildtype (WT) SMN Tudor domain (WT-TD) or its SIM mutants (TD-2VA) of GST-fusion proteins purified from E. Coli expression system and pulled down with Glutathione-sepharose beads. Bound complexes were analyzed by immunoblotting (IB) with the indicated antibodies. The amounts of recombinant proteins were evaluated by Coomassie staining of 10% of the input. c HEK-293T cells were transfected with FLAG-tag SMN wildtype (WT), SIM-less (2 VA), or SMA (E134K) mutants. Cell lysates were prepared for precipitation with anti-FLAG agarose beads. 5% of the input and the immunoprecipitates (FLAG-IP) were analyzed by SDS-PAGE and Western blot with antibodies against the proteins indicated on the right. Naive HEK-293T cells were used as control. * indicate IgG heavy chain. d HEK-293T cells were transfected with FLAG-tag GEMIN5 wildtype (G5-WT) or its non-sumoylatable mutant (G5-2KR). Cell lysates were prepared for precipitation with anti-FLAG agarose beads. Five percent of the input and the immunoprecipitates (FLAG-IP) were analyzed by SDS-PAGE and Western blot with antibodies against the proteins indicated on the right. Naïve HEK-293T cells were used as control. e Representative fluorescent images of HeLa cells transfected with wildtype (WT) or non-sumoylatable (2KR) GEMIN5 and stained with antibodies against SMN (green) and FLAG (GEMIN5 in red), and with DAPI (blue). Scale bar, 20 μm.