Figure 2

The conserved transmembrane and ER luminal loop regions of seipin are required for its interaction with GPAT3. (A) HEK293 cells were transfected with empty vector (−), wild-type Myc-seipin (WT) or mutant seipin lacking the N-terminus (ΔNT), the first transmembrane domain (ΔTM1), the ER luminal loop region (Δloop), the second transmembrane domain (ΔTM2) or the C-terminus (ΔCT) in the absence or presence of FLAG-tagged GPAT3 as indicated. Cell lysates or anti-FLAG immunoprecipitates were separated by SDS-PAGE and immunoblotted with antibodies to FLAG, Myc and calnexin. (B) Quantification analysis of the binding of WT or mutant forms of seipin to FLAG-GPAT3. Data represent means ± SEM (n = 3), normalized to expression levels and expressed as a fold of that observed with wild-type seipin. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001 versus co-immunoprecipitation with wild-type seipin. (C) HEK293 cells were transfected with empty vector (−), N-terminal FLAG-tagged wild-type seipin (WT) or identically-tagged T78A, L91P and A212P forms of seipin in the presence or absence of Myc-tagged GPAT3. Cell lysates or anti-FLAG immunoprecipitates were separated by SDS-PAGE and immunoblotted with antibodies to FLAG, Myc and calnexin. (D) Quantification analysis of the binding of WT or mutant forms of seipin to Myc-GPAT3. Data represent the means ± SEM (n = 3), normalized to expression levels and expressed as a fold of that observed with wild-type seipin. * indicates p < 0.05, ** indicates p < 0.01 versus co-immunoprecipitation with wild-type seipin.