Figure EV5. GIV‐GEM is required for EGF‐triggered secretion of diverse cargo proteins through the Golgi compartment.

• A
  Schematic shows the basis for measuring secretion of transmembrane cargo protein, ts‐VSVG‐eGFP. This temperature‐sensitive mutant VSVG is retained in the ER at 40°C, at the Golgi at 20°C, and moves out of the Golgi to the PM when shifted to 32°C (Gallione & Rose, 1985). When visualized with immunofluorescence under non‐permeabilized conditions, a VSVG‐ectodomain targeting antibody selectively detects PM‐localized cargo, whereas a GFP tag allows the visualization of total VSVG in the cell.
• B, C
  Control (sh Control; top) and GIV‐depleted (shGIV; bottom) Cos7 cells were transfected with tsO45‐VSVG‐GFP and cells were shifted to 40°C for O/N and then incubated at 20°C for 1 h in HEPES buffered serum‐free media followed by temperature shift at 32°C for 15 min in plain DMEM and or containing 50 nM EGF or 10% serum. Coverslips were fixed and stained with VSVG‐ectodomain‐specific monoclonal antibody (red). Representative images are shown in (B). Scale bar = 10 μm. Green fluorescence indicates total VSVG expression whereas red fluorescence shows the surface‐localized pool of VSVG. Bar graphs in (C) display the Red:Green intensity ratio indicative of the fraction VSVG that is secreted to the cell surface. Results are expressed as mean ± SEM; n = 3 biological replicates; P‐values were determined using Mann–Whitney t‐test compared to t0: *P < 0.05.
• D–H
  Control (sh Control) and GIV‐depleted (shGIV) HeLa cells were analyzed for EGF‐stimulated secretion of three soluble cargo proteins, MMP2 (D, E), MMP9 (D, F), and Collagen‐Vii RFP (G, H), as detected from the supernatants at indicated time points after EGF stimulation. Results are expressed as mean ± SEM; n = 3 replicates; P‐values were determined using Mann–Whitney t‐test compared to t0: **P < 0.01; ***P < 0.001. Immunoblots are representative of findings from at least three independent repeats.