1. Reconstitution of GBF1 recruitment to Golgi membranes in a cell-free assay.
Reconstitution of GBF1 recruitment to Golgi membranes in a cell-free assay. (A) WNG membranes recruit GBF1 at physiological temperature, but not on ice. WNG membranes were incubated with GFP-GBF1 NRK cell cytosol at 37°C or on ice for 5 min and then separated into membrane and supernatant fractions by centrifugation. Resulting pellets were separated by SDS-PAGE along with 10% cytosol and WNG controls (WNG Alone). Proteins were transferred to nitrocellulose and incubated with a mouse anti-GBF1 monoclonal antibody and rabbit anti-ManII antibodies, then incubated in donkey anti-mouse Alexa Fluor 750 and donkey anti-rabbit Alexa Fluor 680 secondary antibodies. The resulting immunoblot was then scanned in a Licor Odyssey scanner. A representative blot is displayed. The arrow and the asterisk mark the positions of GFP-tagged and endogenous GBF1, respectively. (B) GFP-GBF1 band intensities were quantified and corrected for the amount of WNG present by normalization to the ManII band intensity as described in Materials and Methods. Error bars are ±s.d. (n=3). Values 0.10, 0.11 and 0.01 were obtained for WNG membranes on ice, and 0.83, 0.59 and 0.59 were obtained for those at 37°.
Douglas Quilty et al. J Cell Sci 2019;132:jcs208199
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