Figure 4. Arf1-binding sites on β1 and γ are required for association of AP-1 to the TGN/endosomes.

(A, B) MDCK-μ1A-HA cells transfected with plasmids encoding β1^WT-GFP or β1^ΔArf1-GFP (A) and γ^WT-GFP or γ^ΔArf1-GFP (B) or GFP (A, B) were subjected to immunoprecipitation (IP) with antibody to GFP followed by SDS-PAGE and immunoblotting with HRP-conjugated antibodies to the HA epitope and GFP. The position of molecular mass markers (in kDa) are indicated on the left. Loading was adjusted to normalize for β1 and γ expression. Assembly of β1 and γ mutants with μ1A-HA was 99±6% and 97±5% of the corresponding wild-type proteins (n=3). (C-H) HeLa cells transfected with plasmids encoding β1^WT-GFP (C-E) or β1^ΔArf1-GFP (F-H) were immunostained for endogenous γ. (I-N) HeLa cells were co-transfected with plasmids encoding γ^WT-mCh (I-K) or γ^ΔArf1-mCh (L-N) together with μ1A-GFP. Nuclei were stained with DAPI. Images were obtained by confocal microscopy. The third image in each row is a merge of images in the green, red and blue channels. Scale bar: 10 μm.