Synchronization assay to study the biosynthetic route consists of the expression of a cDNA with the plasma membrane protein of interest tagged with GFP and fused to four repeats of FM domains, and a furin cleavage site (see Supplementary Figure S4 ). The FM domains are variants of FKBP (FK506-binding protein), which are able to reversibly self-aggregate into homodimers that can disaggregate within minutes after addition of the membrane permeable drug DD solubilizer (Takara Bio Inc., Kusatsu City, Japan, Cat Number: 635053).
Seven DIV culture hippocampal neurons were used for trafficking experiments. Neurons were transfected with TfR-GFP-FM4 or FM4-L1-GFP or FM4-ApoER2-GFP or FM4-p75NTR-GFP plus GalT2-mCherry and BARS mutants or empty vectors. At 12 h post-transfection, the cultures were treated with 100 μg/mL cycloheximide for 1.5 h to prevent newly FM4-GFP-tagged plasma membrane protein synthesis and its entering to the biosynthetic route. Next, neurons were treated with 2.5 µM DD solubilizer (Takara Bio Inc., Cat Number: 635053) and fixed at the indicated times to evaluate the traffic of monitor cargo through the secretory pathway.
Seven DIV culture hippocampal neurons were used for trafficking experiments. Neurons were transfected with TfR-GFP-FM4 or FM4-L1-GFP or FM4-ApoER2-GFP or FM4-p75NTR-GFP plus GalT2-mCherry and BARS mutants or empty vectors. At 12 h post-transfection, the cultures were treated with 100 μg/mL cycloheximide for 1.5 h to prevent newly FM4-GFP-tagged plasma membrane protein synthesis and its entering to the biosynthetic route. Next, neurons were treated with 2.5 µM DD solubilizer (Takara Bio Inc., Cat Number: 635053) and fixed at the indicated times to evaluate the traffic of monitor cargo through the secretory pathway.