JCS028696 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Colocalisation of Flag-VAP and the ER marker calreticulin. These are individual images of Flag or calreticulin from Fig. 1. Scale bar: 10 µm.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Subcellular localisation of VAPs with Sar1 in the absence (−FFAT) or presence (+FFAT) of FFAT. CHO cells were transfected with Flag-tagged VAPs and endogenous Sar1 was detected with anti-Sar1. Scale bar: 10 µm.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Effect of VAPs in the absence (− FFAT) or presence (+ FFAT) of FFAT on microtubule organisation. CHO cells were cotransfected with Flag-tagged VAPs and microtubules were stained with anti-α-tubulin. Scale bar: 10 µm.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Quantitative analysis of VSVG^ts045-GFP morphology. Transfected cells were incubated at 42°C for 6 hours then shifted to 32°C, and fixed with 4% paraformaldehyde at the indicated time (minutes). Cells were scored double-blind for ER phenotype based on colocalisation with calreticulin and for plasma membrane localisation. Approximately 100 cells were counted for each chase period within a set of experiments, and values (mean ± s.e.m., n=3) represent percentage of cells with VSVG-GFP localised to the intracellular compartment at the indicated chase period. Cells were cotransfected with control vector (blue), VAPA-WT (red), VAPA-P56S (green), VAPB-WT (orange) or VAPB-P56S (purple) along with the empty, AAAT or FFAT vector. Cells were scored for localisation at (A) the ER or (B) plasma membrane.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Effect of VAPA on transport of CgB-GFP(S65T). Individual images of localisation of calreticulin and CgB-GFP(S65T) from Fig. 4. Scale bar: 10 µm.

• Movie 1 -

  Movie 1. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell transfected with control vector, with the first frame corresponding to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 2 -

  Movie 2. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell transfected with VAPA-WT, with the first frame corresponding to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 3 -

  Movie 3. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell transfected with VAPA-P56S, with the first frame corresponding to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 4 -

  Movie 4. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell transfected with VAPB-WT, with the first frame corresponding to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 5 -

  Movie 5. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell transfected with VAPB-P56S, with the first frame corresponding to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 6 -

  Movie 6. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell cotransfected with control vector and an FFAT motif, where the first frame corresponds to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 7 -

  Movie 7. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell cotransfected with VAPA-WT and an FFAT motif, where the first frame corresponds to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 8 -

  Movie 8. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell cotransfected with VAPA-P56S and an FFAT motif, where the first frame corresponds to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 9 -

  Movie 9. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell cotransfected with VAPB-WT and an FFAT motif, where the first frame corresponds to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.

• Movie 10 -

  Movie 10. Time-lapse FRAP images collected at 20 second time intervals over the course of a 10 minute chase period. Movie is a representative cell cotransfected with VAPB-P56S and an FFAT motif, where the first frame corresponds to the pre-bleach image followed by the post-bleach recovery interval. The region outlined in the pre-bleach frame corresponds to the bleached area.