Figure 3. SMSr forms SAM-dependent homo-oligomers in the ER.

(a) Detergent extracts of HeLa cells co-transfected with HA-tagged SMSr and V5/His6-tagged SMSr, SMSr∆SAM, SMSr-KKSA or SMSr∆SAM-KKSA were subjected to immunoprecipitation analysis using an anti-V5 antibody. Immunoprecipitates (IP) and total extracts (input) were immunoblotted (IB) using anti-V5 and anti-HA antibodies. An asterisk denotes immunoreactivity with IgG heavy chain. (b) Detergent extracts of HeLa cells co-transfected with HA-tagged SMS1 and V5/His6-tagged SMS1 or SMS1∆SAM were subjected to immunoprecipitation analysis using an anti-V5 antibody. Immunoprecipitates (IP) and total extracts (input) were immunoblotted (IB) using anti-V5 and anti-HA antibodies, as in (a). (c) HeLa cells transfected with empty vector (EV) or V5/His6-tagged SMSr were solubilized using detergent in the presence or absence of 10 mM N-ethyl maleimide (NEM) and subjected to Ni²⁺ -NTA affinity chromatography. Ni²⁺-NTA eluates were incubated in the presence or absence of 100 mM DTT, and immunoblotted (IB) using an anti-V5 antibody, as in (a). (d) HeLa cells transfected with HA-tagged SMSr were treated with chemical crosslinker DSP (0–50 μM, 15 min, RT), solubilized by detergent in the presence of 10 mM NEM and subjected to immunoprecipitation analysis using an anti-HA antibody. Immunoprecipitates were immunoblotted using an anti-HA antibody. (e) Yeast cells transfected with empty vector (EV) or V5/His6-tagged SMSr were treated with DSP as in (d), lysed and subjected to immunoblot analysis using an anti-V5 antibody. (f) HeLa cells transfected with empty vector (EV), V5/His6-tagged SMSr or SMSr∆SAM were incubated in the presence or absence of DSP (50 μM, 15 min, RT), solubilized with detergent in the presence of 10 mM NEM and subjected to Ni²⁺-NTA affinity chromatography. Ni²⁺-NTA eluates were immunoblotted using an anti-V5 antibody. Uncropped images of blots are provided in Supplementary Fig. S1.