Figure 4. Overexpression of VAPB and/or PTPIP51 increases ER–mitochondria associations in CV-1 cells.

Confocal immunostaining of CV-1 cells transfected with control ECFP vector (CTRL; blue), ECFP-VAPB (VAPB; blue), PTPIP51-HA (PTPIP51; blue) or ECFP-VAPB+PTPIP51-HA (VAPB+PTPIP51) and immunostained for ER (using mouse PDI antibody) and mitochondria (using rabbit TOM-20) as indicated. In VAPB+PTPIP51 co-transfected cells, only ECFP-VAPB (blue) labelling is shown but duplicate coverslips were immunostained for ECFP-VAPB and PTPIP51-HA, and this demonstrated that ~95% of cells expressed both transfected proteins. These data are consistent with many previous reports, which show that most co-transfected cells express both plasmids (for example, ref. 59) Merge is of ER and mitochondria labelling only; the zoom view shows co-localized pixels. Scale bar, 20 μm. Bar chart shows intensity correlation quotient (ICQ) values (with ECFP control represented as 100%) in the different transfections. Data were analysed by one-way analysis of variance with Tukey’s post hoc test. N=34–52 cells; error bars are s.e.m.; ***P<0.001.