Figure 3. Src activates the ARF-GEF GBF1.

(A) Representative SDS-PAGE analysis of Arf1-GTP levels in HEK-IS cells expressing GFP or GFP-GBF1. GGA pulldown was performed as in Figure 2E. (B) Quantification of Arf1-GTP levels in three independent experiments in (A). (C) SDS-PAGE analysis of Arf1-GTP levels in HEK-IS cells treated with siGBF1 and siNT siRNA. (D) Quantification of the Arf1-GTP levels in three independent experiments in (C). (E) SDS-PAGE analysis of cytoplasmic and membrane levels of GBF1 after imidazole (imdz) stimulation. (F) Quantification of two independent experiments shown in (E). Values presented were normalized to untreated cells (0 hr).(G) Still images of the time-lapse movie of GBF1-GFP in HeLa-IS cells stimulated with 5 mM imdz. Scale bar: 10 μm. (H) Quantification of the ratio of Golgi to total cytoplasmic levels of GBF1 before and after imdz treatment in time lapse shown in (G). (I) SDS-PAGE analysis of the levels of Arf1-V5 bound to GFP or GFP-GBF1 immunoprecipitation (IP) from cells expressing inactive SrcKM or active SrcEG in an in vitro binding assay. Two experimental replicates were tested and quantified in Figure 3—figure supplement 1E.

Figure 3—figure supplement 1. GBF1 is required for GALNT tubule formation.

(A) Helix pomatia lectin (HPL) staining of HeLa-IS stable cell line treated with siRNA targeting GBF1 before and after 4 hr of imidazole (imdz) treatment. siNT refers to non-targeting siRNA, and siGALNT1 + T2 refers to co-transfection of GALNT1 and GALNT2 siRNAs. Images were acquired under constant acquisition settings using an automated confocal microscope. Scale bar: 50 μm. (B) Quantification of HPL staining intensity per cell normalized to the respective untreated cells (0 hr) for each siRNA treatment. (C) Immunoblot analysis of the levels of Tn-modified endoplasmic reticulum (ER)-resident PDIA3 from Vicia villosa lectin (VVL) immunoprecipitation (IP) in HEK-IS cell line upon GBF1 siRNA knockdown. Cells were untreated or treated with 5 mM imdz for 6 hr. (D) Schematic illustrating the workflow of the in vitro Arf1 binding assay. (E) Quantification of the levels of bound Arf1-V5 to GFP and GFP-GBF1 (WT) IP from cells expressing inactive SrcKM or active SrcEG in the in vitro binding assay shown in Figure 3G. Results representative of two experimental replicates. (F) SDS-PAGE analysis of the levels of recombinant protein Arf1-del17-His bound to GFP-GBF1 IP from inactive SrcKM or active SrcEG-expressing cells in an in vitro binding assay. (G) Images from time-lapse imaging of GALNT2-GFP in HeLa-IS cells that were either treated with siRNA targeting GBF1 (siGBF1) or siNT stimulated with 5 mM imdz. (See Figure 3—videos 1 and 2 for time-lapse movies.) Scale bar: 5 μm. (H) Quantification of the number of tubules observed in the first 30 min upon imidazole treatment. Values on graphs indicate the mean ± SD. Statistical significance (p) was measured by two-tailed paired t-test. *p<0.05 and **p<0.01 relative to untreated (0 hr) or GFP-expressing cells. NS, nonsignificant.

Figure 3—video 1. Video of tubule formation in siNT-treated HeLa-IS cells expressing GALNT2-GFP stimulated with 5 mM imidazole (imdz).

Download video file ^{(22.9MB, mp4)}

Scale bar: 5 μm.

Figure 3—video 2. Video of tubule formation in siGBF1-treated HeLa-IS cells expressing GALNT2-GFP stimulated with 5 mM imidazole (imdz).

Download video file ^{(21.8MB, mp4)}

Scale bar: 10 μm.