Figure 5. SSTR2 fractionates with a... | Rockefeller University Press

Figure 5.

$SSTR2 fractionates with a syntaxin-6 compartment distinct from the TGN. (A) Immunoisolation of syntaxin-6–positive vesicles. HEK-293 cells were transfected with T7-SSTR2 alone or in combination with HA-EGFP-syntaxin-6. Cell lysates were prepared in detergent free buffer, and HA-EGFP-syntaxin-6 structures were isolated using anti-HA magnetic beads. The proteins were separated by SDS-PAGE and probed by immunoblot using antibodies recognizing the indicated proteins. The migration of molecular weight markers in kilodaltons is indicated. IP, immunoprecipitate. (B) Sucrose gradient separation of the syntaxin-6 compartment. 2 ml of AtT20 postnuclear supernatant collected from three 10-cm dishes was fractionated on a 10-ml continuous 10–40% (wt/vol) sucrose density gradient. Fractions (500 µl) were harvested, beginning at the top of the gradient. Equal volumes from every other fraction were separated by SDS-PAGE and processed for immunoblot using antibodies against recognizing PIST, TGN38, GM130, and syntaxin-6. n = 4. The migration of molecular weight markers in kilodaltons is indicated.$

SSTR2 fractionates with a syntaxin-6 compartment distinct from the TGN. (A) Immunoisolation of syntaxin-6–positive vesicles. HEK-293 cells were transfected with T7-SSTR2 alone or in combination with HA-EGFP-syntaxin-6. Cell lysates were prepared in detergent free buffer, and HA-EGFP-syntaxin-6 structures were isolated using anti-HA magnetic beads. The proteins were separated by SDS-PAGE and probed by immunoblot using antibodies recognizing the indicated proteins. The migration of molecular weight markers in kilodaltons is indicated. IP, immunoprecipitate. (B) Sucrose gradient separation of the syntaxin-6 compartment. 2 ml of AtT20 postnuclear supernatant collected from three 10-cm dishes was fractionated on a 10-ml continuous 10–40% (wt/vol) sucrose density gradient. Fractions (500 µl) were harvested, beginning at the top of the gradient. Equal volumes from every other fraction were separated by SDS-PAGE and processed for immunoblot using antibodies against recognizing PIST, TGN38, GM130, and syntaxin-6. n = 4. The migration of molecular weight markers in kilodaltons is indicated.