(A) Co-immunoprecipitation by FLAG and hemagglutinin (HA) antibody of overexpressed FLAG-tagged GPAT3 or GPAT4 and HA-tagged SEIPIN, respectively, from transfected MEF cell lysates.
(B and C) Immunoblotting for overexpressed HA-tagged mutant or wild-type SEIPIN and FLAG-tagged GPAT3 and GPAT4, respectively, after HA-immunoprecipitation from 3T3-L1 cells. See also Figure S2.
(D) Endogenous GPAT4 and GPAT3 proteins co-immunoprecipitate with overexpressed SEIPIN from 3T3-L1 adipocytes stably transfected with HA- or FLAG-SEIPIN.
(E) 3T3-L1 adipocytes expressing FLAG-SEIPIN, FLAG-tagged biotin ligase BirA*, or FLAG-tagged BirA*-SEIPIN were treated with 50 μM biotin for 24 hr. The biotinylated proteins were pulled down using streptavidin-conjugated agarose from biological duplicates. Elutes were subjected to western blot analysis. All experiments were performed in triplicate.