JCS03413 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Salt and Triton extraction of synapsin from microsomes. LDM fractions were prepared from (A) mouse brain (Br) or (B) adipocytes (1^o and L1). The pellets were resuspended in HEPES-buffered sucrose (HES), HEPES-buffered salt (NaCl, 200 mM), HES with Triton X-100 (TX100; 1%) or NaCl with TX100, incubated for 15 minutes on ice, then centrifuged at 150,000 g. Equal amounts (% of total) of the soluble (S) and particulate (P) fractions were analyzed by western blotting with antibodies against synapsin IIb (Syn). The release of the transmembrane vesicle proteins Glut4 and the transferrin receptor (TfR) from L1 adipocyte LDM was also analyzed. Synapsin IIb is released into the soluble fraction by salt treatment, but remains associated with the particulate fraction after addition of TX100. By contrast, Glut4 and the transferrin receptor remain associated with the particulate fraction after addition of salt, and are largely released into the soluble fraction after treatment with TX100.

• Supplemental Figure 2 -

  Fig. S2. Expression of HA-Glut4-GFP and Flag-synapsin. Fibroblasts or adipocytes were infected with virus expressing HA-Glut4-GFP alone (G4), or co-expressing WT-synapsin IIb (G4/S) or S10A synapsin IIb (G4/S10A). Uninfected cells (NV) were also analyzed. (A,B) Similar levels of HA-Glut4/GFP were expressed from all viruses. (A) Western blotting for HA-Glut4/GFP (anti-HA) in lysates; (B) flow cytometric analysis of relative HA-Glut4/GFP (GFP) expression in adipocytes. MFI: mean fluorescence intensity ± s.d. (C,D) The level of expression of HA-Glut4/GFP was relatively low, as no increase in total Glut4 was detected in adipocytes expressing this construct. (C) Flow cytometric analysis of relative Glut4 expression in adipocytes. Adipocytes were detached from the plate with collagenase, then fixed, permeabilized, and labeled with anti-Glut4 antibody (α-Glut4; detected with Alexa Fluor 647-APC). The antibody used to detect total Glut4 recognizes both endogenous Glut4 (in uninfected adipocytes) and the HA-Glut4/GFP (in infected fibroblasts, uninfected fibroblasts have no endogenous Glut4). (D) Two-dimensional histograms of total Glut4 (α-Glut4; detected with Alexa Fluor 647-APC) vs GFP levels in infected (G4) and uninfected (NV) adipocytes and fibroblasts. (E,F) Similar levels of synapsin were expressed from all viruses, at approximately the same levels as in the brain, and all cells positive for GFP fluorescence (HA-Glut4/GFP) were also positive for Flag labeling in cells expressing WT- or S10A synapsin. (E) Western blotting for Flag-synapsin or total synapsin (Br, brain). Equal amounts of protein were loaded in all samples. (F) Flow cytometric analysis of anti-Flag labeling (detected with Alexa Fluor 647-APC) vs GFP fluorescence (cells positive for both HA-Glut4-GFP and Flag-synapsin are in the upper right hand quandrant). (G) The collagenase used to detach the labeled cells from the culture dishes does not interfere with anti-HA binding. Infected fibroblasts were labeled with Alexa Fluor 647-conjugated anti-HA antibody on ice for 2 hours. The labeled cells were detached from the plate using collagenase (5 minutes, 37°C) or gentle pipetting and analyzed by flow cytometry. There are equal levels of antibody bound to cells using either method to suspend the cells. Very little non-specific binding was observed in the flow experiments as detected in uninfected cells. The signal to noise ratio was greater than 10 for GFP and at least 2-3 for the PC7-anti-HA (basal samples; insulin samples were six- to sevenfold higher).

• Supplemental Figure 3 -

  Fig. S3. Synapsin IIb and Glut4 are co-localized. Individual z-stack images (0.5 μm slices through the center of the cell) used to create the maximum projections shown in Fig. 3. Note that co-localization of Flag-synapsin with a perinuclear cluster of Glut4 vesicles is observed in multiple sequential sections. G4/GFP, HA-Glut4/GFP; Flag-Syn, WT-Flag-synapsin IIb (G4/S), S10A Flag-synapsin IIb (G4/S10A) or S10D Flag-synapsin IIb (G4/S10D).