Figure 5.

Deficiency of Cog8 affects the subcellular distribution of Stx6 and VAMP4 and impairs their assembly into SNARE complex. A) The Golgi localization of Stx6 or VAMP4 (red) in control or Cog8-deficient fibroblasts was determined by co-immunostaining with the Golgi marker p115 (green) and confocal microscopy analysis. In the patient’s cells, the Golgi localization of VAMP4 was reduced concomitantly with an increase in its cytosolic haze-like staining. In addition, a pool of Stx6 accumulated in punctate structures. Bars: 10 µm. B) HeLa cells were transiently transfected with a control or Cog8 shRNA construct, and 72 h later the cells were fixed and double immunostained with the indicated antibodies. The localization of Stx6 and VAMP4 (red) to the Golgi membranes was determined by co-immunostaining with p115 (green). In Cog8-depleted HeLa cells, VAMP4 was completely dispersed from the Golgi, while Stx6 displayed a weak residual Golgi staining. Bars: 10 µm. C) Cog8- and Cog7-deficient fibroblasts obtained from human patients (left panels) or Cog8-depleted HeLa cells (right panels) with their corresponding controls were pretreated with NEM, solubilized and subjected to IP with anti-Stx6 antibody. The presence of the indicated SNARE proteins in the immunocomplexes was determined by WB using the corresponding antibodies.