FIGURE 3.

Co-immunoprecipitation of DGAT2 and MGAT2 from HEK293T cells. A, FL-MGAT2 was co-expressed with either myc-DGAT2 or untagged DGAT2 in HEK-293T cells. MGAT2 was immunoprecipitated with anti-FLAG agarose from detergent solubilized material. Immunoprecipitates (IP) were separated by SDS-PAGE and were then probed with anti-FLAG and anti-DGAT2. Immunoprecipitates were also probed with anti-PDI (Assay Designs) and anti-HSP70 (Thermo Scientific) antibodies as controls for nonspecific interactions. B, reciprocal co-immunoprecipitation of MGAT2 and DGAT2. FL-DGAT2 was co-expressed with myc-MGAT2 in HEK-293T cells. DGAT2 was immunoprecipitated with anti-FLAG-agarose from detergent solubilized material. Immunoprecipitates were separated by SDS-PAGE and were then probed with anti-FLAG and anti-myc antibodies. This experiment was repeated once with similar results. C, Lipin1 interacts weakly with both DGAT1 and DGAT2. FL-Lipin1 was co-expressed with either myc-DGAT1 or myc-DGAT2 in HEK-293T cells. FL-Lipin1 was immunoprecipitated with anti-FLAG agarose from detergent-solubilized material. Immunoprecipitates were separated by SDS-PAGE and were then probed with anti-myc. To visualize DGAT1 and DGAT2, PVDF membranes were exposed to film for extended periods of time. D, interaction of DGAT2 and MGAT2 was detected in situ using a proximity ligation assay. COS-7 cells expressing either FL-DGAT2, myc-MGAT2, or both together were stained with mouse anti-FLAG and rabbit anti-myc antibodies. Interaction signals (red) were detected using a Duolink detection kit. Nuclei were stained with DAPI (blue), and lipid droplets were stained with BODIPY 493/503 (green). Scale bar, 10 μm.