Figure 2: Impact of CRN7 deletion on Golgi architecture.
From: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology
(a) Immunoblot analysis of WT and KO fibroblast lysates. CRN7 detected with mAb K37-142-1. GAPDH, loading control. (b) Detection of the Golgi apparatus in the fibroblasts using mAb 58 K-9 against the 58K Golgi membrane protein (green); nuclei stained with DAPI (blue). White asterisk, compact Golgi, yellow asterisk, dispersed Golgi. Scale bar = 7.5 μm. (c) Analysis of Golgi dispersal for 400 cells. Percentages of WT and KO cells with a fragmented or compact Golgi represented as stacked columns (n = 200 cells each, 2 independent experiments; *P < 0.05). (d) WT (left) and KO (right) primary cells transiently transfected with GFP-GalT were photobleached, and FRAP was measured over time. The curves show the normalized FRAP kinetics over time. Fluorescence recovery in bleached areas was monitored every 5 s. Curves of fluorescence intensities were normalized to nonbleached area and the background. Shown is mean ± SEM of 10 cells per condition. Mf and t1/2 were determined by fitting the curves to a one-phase exponential equation. (e) Immunoblot analysis of lysates from KO fibroblasts expressing GFP-CRN7 (rescue). Detection using mAb K37-142-1. (f) KO and rescue cells stained for Golgi (red) and rescue cells are green. The Golgi has been marked with asterisks as in (b). Scale bar = 10 μm. (g) 300 cells scored for their Golgi phenotype. Percentages of WT and KO cells with a fragmented or compact Golgi are illustrated as in (c) (n = 150 cells each, 2 independent experiments; *P < 0.05). Except FRAP all data shown as mean ± SD.
