Figure 6. Proximity of GCC185 N- and C-termini on the Golgi requires putative hinge sequences.
(A) Top: Proximity ligation in HeLa cells using indicated antibodies; nuclear staining is shown below. (B) Quantitation of proximity ligation using CellProfiler; N = 168, 94 or 46, left to right (p<0.0001 by two-tailed Student’s t-Test). (C) Bottom: Proximity ligation using mouse-anti-GFP and rabbit-anti-GCC185 C-terminus antibodies in GFP-GCC185 or GFP-GCC185-Δhinge-transfected HeLa cells. Top, GFP-GCC185 expression in the same cells. Bar, 20 μm. (D) Quantitation of proximity ligation versus GFP-protein expression levels using CellProfiler. More than 300 objects were measured for each condition (p<0.001 by two-tailed Student’s t-Test), pooled from two independent experiments. (E) Immunoblot test of antibody specificity using purified GCC185 domains. Upper panel: 10 ng GST-1–358 and His-1342–1684 were detected with (left) mouse anti-GCC185-N-term or (right) rabbit anti-GCC185-C-term antibodies. Lower panel: blots were re-probed using mouse anti-His (left) or rabbit anti-GST (right) antibodies to detect the corresponding antigens.