Figure 5.
Golgi bypass early delivery of RUSH-α5 requires the integrin-α5 PDZ-binding motif. (A and B) Quantification of relative RUSH-α5 recruitment to protruding areas (A) or adhesions (B) in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times with or without Golgicide A (10 µM). (C) Representative immunoblot of GFP pulldowns from RUSH-α5 or GFP control transfected cells plated on FN and probed for GFP and endogenous GRASP65. N = 3 independent experiments. (D) Amino acid sequence of the integrin α5 tail highlighting the canonical PDZ-binding motif (SDA) and the two proline residues critical for the formation of the non-canonical PDZ-binding motif. The mutations of these sites used in our experiments are indicated below. (E) Representative streptavidin pulldowns of the indicated biotinylated recombinant integrin peptides incubated with cell lysates collected from CHO cells overexpressing GFP-GRASP65. A representative immunoblot probed for GRASP65 (note, two bands are present in the lysate: upper, GFP-GRASP65; lower, endogenous GRASP65; GFP-GRASP65 is apparent in the pulldown). N = 3 independent experiments. (F) Quantification of RUSH-α5 or RUSH-α5 ΔSDA recruitment to adhesions in U2OS cells ± biotin treatment for the indicated times. All data are mean ± SD. (A and B) One-way ANOVA with Tukey’s multiple comparison test for comparing time points, one-way ANOVA with Holm-Šídák’s multiple comparisons test for comparing untreated and Golgicide A, data distribution was assumed to be normal but this was not formally tested. (A)N = 26 cells RUSH-α5, N = 22 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (B)N = 24 cells RUSH-α5, N = 27 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (F) One sample t test to compare time points with T = 0, ordinary one-way ANOVA with Holm-Šídák’s multiple comparisons test to compare RUSH-α5 and RUSH-α5 ΔSDA, data distribution was assumed to be normal but this was not formally tested. N = 23 cells RUSH-α5, N = 23 cells RUSH-α5 ΔSDA, pooled from two independent experiments. Source data are available for this figure: SourceData F5.

Golgi bypass early delivery of RUSH-α5 requires the integrin-α5 PDZ-binding motif. (A and B) Quantification of relative RUSH-α5 recruitment to protruding areas (A) or adhesions (B) in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times with or without Golgicide A (10 µM). (C) Representative immunoblot of GFP pulldowns from RUSH-α5 or GFP control transfected cells plated on FN and probed for GFP and endogenous GRASP65. N = 3 independent experiments. (D) Amino acid sequence of the integrin α5 tail highlighting the canonical PDZ-binding motif (SDA) and the two proline residues critical for the formation of the non-canonical PDZ-binding motif. The mutations of these sites used in our experiments are indicated below. (E) Representative streptavidin pulldowns of the indicated biotinylated recombinant integrin peptides incubated with cell lysates collected from CHO cells overexpressing GFP-GRASP65. A representative immunoblot probed for GRASP65 (note, two bands are present in the lysate: upper, GFP-GRASP65; lower, endogenous GRASP65; GFP-GRASP65 is apparent in the pulldown). N = 3 independent experiments. (F) Quantification of RUSH-α5 or RUSH-α5 ΔSDA recruitment to adhesions in U2OS cells ± biotin treatment for the indicated times. All data are mean ± SD. (A and B) One-way ANOVA with Tukey’s multiple comparison test for comparing time points, one-way ANOVA with Holm-Šídák’s multiple comparisons test for comparing untreated and Golgicide A, data distribution was assumed to be normal but this was not formally tested. (A)N = 26 cells RUSH-α5, N = 22 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (B)N = 24 cells RUSH-α5, N = 27 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (F) One sample t test to compare time points with T = 0, ordinary one-way ANOVA with Holm-Šídák’s multiple comparisons test to compare RUSH-α5 and RUSH-α5 ΔSDA, data distribution was assumed to be normal but this was not formally tested. N = 23 cells RUSH-α5, N = 23 cells RUSH-α5 ΔSDA, pooled from two independent experiments. Source data are available for this figure: SourceData F5.

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