Home Abstract Introduction
Immunofluorescence of VAPs and Calreticulin Time Course Immunofluorescence of VSVG with relationship to the FFAT motif Immunofluorescence of Calreticulin and Chromogranin B Time Course Immunofluorescence of properly folded VSVG Quantitative Western Immunoblot of VSVG for ER budding Western Blots and Quantification of Microtubule binding of FFAT and AAAT
Implications Support

Translation of Journal of Cell Science

Prosser, D. C., et al.
"FFAT Rescues VAPA-Mediated Inhibition of ER-to-Golgi
Transport and VAPB-Mediated ER Aggregation."
Journal of Cell Science, vol. 121, no. 18, 19 June 2008, pp. 3052-3061.,
doi:10.1242/jcs.028696.

(Translated by Sohail Syed)

Time Course Immunofluorescence of properly folded VSVG

Motivation

The motivation behind this experiment was to test whether proteins are still being folded properly in the WT VAPA and WT VAPB. and whether their diffusion changes due to VAPA/VAPB.

Procedure

GFP and I14 anti-bodies are used as they bind to properly folded VSVG

Steps for Time Course Immunofluorescence

    1. Cells are incubated at 42C for 6 hours to trap VSVG

    2. Shift temperature to 32C

    3. Flag anti-bodies are attached to the beginning of the proteins of interest (GFP and I14)

    4. Image by confocal microscopy at 0 min and 30 min

Results

Trapped folded VSVG is still present in the ER for VAPA WT

Allows us to rule out VAPs effect on protein folding

Normal folding leaving the ER/Golgi in control and VAPB WT