Supplementary Figure 1: Expression and interaction analyses of candidate cargo receptors.
From: CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis
(a), ER localization of YFP-tagged candidate cargo receptors. Confocal microscopy analysis of Y1- or Y2-tagged ER candidate proteins (green) shows co-localization of all of the constructs with the ER marker, KDEL (red), and no discernible alteration of ER morphology following protein overexpression. Scale bar: 20 μm. (b), Shown is a BiFC assay of candidate cargo receptors tagged with Y1 and five pools of Y2-tagged lysosomal enzymes, each pool containing 10 different enzymes. Control experiments (CTRL) were performed by co-transfecting Y1-tagged candidates with pools of Y1-tagged lysosomal enzymes. Scale bar: 200 μm. (c), Details of the BiFC-flow cytometry analysis. CLN8 proteins form homodimers and this property has been used to set up the BiFC-flow cytometry assay. Shown are examples of histogram representations of BiFC data for CLN8 Y1/Y1 and Y1/Y2 controls (left), CLN8/TPP1 interaction (center), and CLN8/PRCP lack of interaction (right). In all analyses, cells were co-transfected with a Ruby plasmid (red fluorescence), used to normalize BiFC readings for transfection efficiency. Images shown in (a), (b) and (c) are representative of n = 3 independent experiments
