Journal: eLife

Article Title: Src activates retrograde membrane traffic through phosphorylation of GBF1

doi: 10.7554/eLife.68678

Figure Lengend Snippet: ( A ) Representative images of Helix pomatia lectin (HPL) staining in HeLa-IS treated with various siRNA 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. Scale bar: 50 μm. ( B ) Quantification of HPL staining intensity per cell normalized to the respective untreated cells (0 hr) for each siRNA treatment. Three replicate wells per experiment were measured. ( C ) Representative images of GALNT2-expressing HeLa-IS cells stained for Arf1 before and after 10 min of stimulation with 5 mM imdz. Images were acquired at ×100 magnification. Scale bar: 5 μm. ( D ) SDS-PAGE analysis of cytoplasmic and membrane levels of Arf1 after imdz stimulation. CANX refers to blotting for endoplasmic reticulum (ER)-resident Calnexin. The blots were generated with the same exposure and repeated twice. ( E ) SDS-PAGE analysis of GTP-loaded Arf1 after pulldown with GGA3 beads after imdz treatment in HEK-IS cells. ( F ) Quantification of Arf1-GTP levels in ( E ). Two experimental replicates were measured and values were normalized to untreated cells (0 hr). Values on graphs indicate the mean ± SD. Statistical significance (p) was measured by two-tailed paired t -test. *p<0.05, **p<0.01, ***p<0.001 relative to untreated cells. NS, nonsignificant.

Article Snippet: GGA3 PBD agarose beads were purchased from Cell Biolabs, Inc (San Diego, CA).

Techniques: Staining, Cotransfection, Expressing, SDS Page, Membrane, Generated, Two Tailed Test

Journal: eLife

Article Title: Src activates retrograde membrane traffic through phosphorylation of GBF1

doi: 10.7554/eLife.68678

Figure Lengend Snippet: ( A ) Additional representative images of Arf1 on GALNT2 tubules emanating from the Golgi upon 10 min stimulation of 20 mM imidazole (imdz). Images were acquired at ×100 magnification under Immersol oil. Scale bar: 5 μm. ( B ) SDS-PAGE analysis of the levels of Arf1-GTP immunoprecipitation (IP) using GGA3 beads in HEK293T cells expressing empty mCherry vector, SrcKM and SrcEG mutants. ( C ) Quantification of the levels of Arf1-GTP in ( B ). Three experimental replicates were measured. ( D ) SDS-PAGE analysis of total lysate (L), cytoplasmic (C), and membrane (M) levels of various proteins after subcellular fractionation. Endoplasmic reticulum (ER)-resident Calnexin (CANX), Golgi marker GM130 as well as cytoplasmic actin and MAP kinase ERK8 are shown. ( E ) siRNA knockdown efficiencies of various proteins assayed. siNT refers to non-targeting siRNA. Values on graphs indicate the mean ± SD. Statistical significance (p) was measured by two-tailed paired t -test. *p<0.05 and **p<0.001 relative to untreated cells. NS, nonsignificant.

Article Snippet: GGA3 PBD agarose beads were purchased from Cell Biolabs, Inc (San Diego, CA).

Techniques: SDS Page, Immunoprecipitation, Expressing, Plasmid Preparation, Membrane, Fractionation, Marker, Knockdown, Two Tailed Test