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)

Immunofluorescence of Calreticulin and Chromogranin B

Motivation

The motivation behind this experiment was to check to see if soluble proteins still diffuse through the ER/Golgi/Membrane and whether they are affected by the over-expression of VAPA/VAPB

Procedure

Steps for Immunofluorescence of Calreticulin and Chromogranin B

    1. Flag anti-bodies are attached to the beginning of the proteins of interest (Calreticulin and Chromogranin B)

    2. Incubate cells at 15C for 2 hours to trap Chromogranin B in the ER

    3. Shift temperature to 37C

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

Results

Control

  • Normal diffusion of soluble proteins

VAPA WT

  • Normal diffusion of soluble proteins

VAPA P56S

  • Normal diffusion of soluble proteins

VAPB WT

  • Normal diffusion of soluble proteins

VAPB P56S

  • Normal diffusion of soluble proteins

Expression of VAPs does not affect soluble proteins but does affect transmembrane proteins such as VSVG

Immobile obstacles do not affect movement of soluble proteins

The structure of the ER does not affect soluble protein movement