Figure 3. Localization of DENND1A is clathrin dependent. (A) HeLa cells expressing EGFP-tagged DENND1A (green) were fixed and then stained with antibodies to clathrin heavy chain, and the AP-1, AP-2, and AP-3 clathrin adaptor complexes (red). DNA was stained with DAPI (blue). (B) DENND1A and DENND1B complexes were analyzed by mass spectrometry. The proteins scored highest by the Sequest search algorithm are listed in the table. (C) HeLa cells were transfected with constructs encoding FLAG-tagged DENND1A, DENND1B-L, DENND1B-S, and DENND1C for 48 h. The cells were washed from the dish using PBS with 1 mM EDTA, and the cell pellets were lysed for 20 min on ice in 1 ml cell of lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, and protease inhibitors cocktails). The FLAG-tagged proteins were immunoprecipitated from the clarified lysate using 20 µl of anti-FLAG M2 affinity gel (Sigma-Aldrich) for 4 h at 4°C. The pellet was washed three times in 1 ml of cell lysis buffer, and bound proteins were eluted with 1 ml of 200 µg/ml FLAG peptide in TBS and then precipitated for 60 min on ice using 10% trichloroacetic acid. The FLAG-tagged DENND1A, DENND1B-L, DENND1B-S, and DENND1C complexes were analyzed by SDS-PAGE on 4–12% gradient gels and Coomassie blue staining, or Western blotted for clathrin heavy chain (CHC) and the AP-1 and AP-2 clathrin adaptors on 10% gels. Asterisks mark proteins that nonspecifically bind to FLAG-agarose and were found in negative control conditions. Molecular mass standards are indicated in kilodaltons. (D) HeLa cells expressing EGFP-tagged DENND1A (green) were transfected for 72 h with siRNA duplexes targeting the clathrin heavy chain, fixed, and then stained with antibodies for clathrin and the AP-2 clathrin adaptor (red). DNA was stained with DAPI (blue). Enlargements are shown to the right to more clearly demonstrate the overlap between DENND1A (green), and clathrin or AP2 (red) in control cells, and the loss of punctate DENND1 staining after clathrin heavy chain depletion. Bars, 10 µm.
Figure 3.

Localization of DENND1A is clathrin dependent. (A) HeLa cells expressing EGFP-tagged DENND1A (green) were fixed and then stained with antibodies to clathrin heavy chain, and the AP-1, AP-2, and AP-3 clathrin adaptor complexes (red). DNA was stained with DAPI (blue). (B) DENND1A and DENND1B complexes were analyzed by mass spectrometry. The proteins scored highest by the Sequest search algorithm are listed in the table. (C) HeLa cells were transfected with constructs encoding FLAG-tagged DENND1A, DENND1B-L, DENND1B-S, and DENND1C for 48 h. The cells were washed from the dish using PBS with 1 mM EDTA, and the cell pellets were lysed for 20 min on ice in 1 ml cell of lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, and protease inhibitors cocktails). The FLAG-tagged proteins were immunoprecipitated from the clarified lysate using 20 µl of anti-FLAG M2 affinity gel (Sigma-Aldrich) for 4 h at 4°C. The pellet was washed three times in 1 ml of cell lysis buffer, and bound proteins were eluted with 1 ml of 200 µg/ml FLAG peptide in TBS and then precipitated for 60 min on ice using 10% trichloroacetic acid. The FLAG-tagged DENND1A, DENND1B-L, DENND1B-S, and DENND1C complexes were analyzed by SDS-PAGE on 4–12% gradient gels and Coomassie blue staining, or Western blotted for clathrin heavy chain (CHC) and the AP-1 and AP-2 clathrin adaptors on 10% gels. Asterisks mark proteins that nonspecifically bind to FLAG-agarose and were found in negative control conditions. Molecular mass standards are indicated in kilodaltons. (D) HeLa cells expressing EGFP-tagged DENND1A (green) were transfected for 72 h with siRNA duplexes targeting the clathrin heavy chain, fixed, and then stained with antibodies for clathrin and the AP-2 clathrin adaptor (red). DNA was stained with DAPI (blue). Enlargements are shown to the right to more clearly demonstrate the overlap between DENND1A (green), and clathrin or AP2 (red) in control cells, and the loss of punctate DENND1 staining after clathrin heavy chain depletion. Bars, 10 µm.

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