Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin
Fig 7
Premature calnexin palmitoylation is prevented by serine phosphorylation.
A: Hela cells were transfected 48 hrs with Calnexin-GFP, GFP-DHHC6 or GFP-Climp63. Cells were submitted to FRAP analysis (see Material and Methods). B-E: HeLa cells were transfected for 24h with calnexin-WT-HA, calnexin-S3A-HA, calnexin-AA-HA or calnexin-AA-S3A-HA. C: Cells were incubated with 3H-palmitic acid for 2h, washed prior to immunoprecipitation using anti-HA antibodies. Immunoprecipitates were split into two, run on SDS-PAGE and analyzed either by autoradiography (3H-palmitate) or Western blotting (anti-HA or anti phospho-calnexin). Autoradiograms were quantified using the Typhoon Imager (Image QuantTool, GE healthcare). Errors correspond to standard deviations (n = 3). D: Cells were incubated with 3H-palmitic acid for different hours at 37°C, washed prior to immunoprecipitation using anti-HA antibodies. Immunoprecipitates were split into two, run on SDS-PAGE and analyzed either by autoradiography (3H-palmitate) or Western blotting and autoradiograms were quantified using the Typhoon Imager (Image QuantTool, GE healthcare). E: Cells were incubated 20 min pulse at 37°C with 35S-methionine/cysteine, washed and further incubated for different times at 37°C. Calnexin were immunoprecipitated and analyzed by SDS-PAGE. Autoradiography and western blotting were quantified using the Typhoon Imager (Image QuantTool, GE healthcare).