Figure 3. Depletion of the TRC40 pathway receptor results in mislocalization of a subset of TA-proteins in vivo.

(a) Ventricular cardiomyocytes from eight-week old MerCreMer- (control) and MerCreMer+ (KO) Wrb^fl/fl littermates two weeks after tamoxifen induction were isolated and subcellular localization of selected TA proteins (EMD, Stx5, Stx6, Stx8, Sec61β) was analysed by indirect immunofluorescence. Images were acquired with a confocal microscope. The multi-spanning membrane protein LBR served as control. Scale bar: 20 μm. (b) Quantification of mislocalization phenotype. For each protein, 22 to 98 cells isolated from 4 to 8 animals were examined. The scoring was performed blindly by three investigators using an image shuffling pipeline and automated genotype/phenotype decoder. The following criteria were applied to assign an “altered” phenotype: for Stx5, Stx6 and Stx8 loss of staining at membraneous structures resembling Golgi or endosomes; for Sec61β loss of staining at cellular striations resembling the sarcoplasmic reticulum; for emerin and LBR loss of staining at the nuclear rim and sarcoplasmic reticulum striations. (c) Isolated hepatocytes from six-week old animals were immunostained for either Stx5 or Stx8 and images were acquired with a confocal microscope. GM130 was used as a Golgi marker. Scale bar: 20 μm. (d) Live-cell microscopy of wild type and get1/get2 yeast cells expressing GFP-Sed5 or GFP-Syn8. Scale bar: 5 μm.