The Journal of Neuroscience, March 28, 2007, ():
Rho Regulates Membrane Transport in the Endocytic Pathway to Control Plasma Membrane Specialization in Oligodendroglial Cells
J. Neurosci. Kippert et al.
27: 3560
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Figure S1
Neurons reduce the uptake of Cholera toxin-FITC in Oli-neu cells
A, Oli-neu cells were pretreated for 4 h with conditioned neuronal medium (cnm) or left untreated (ctrl) and ChxB conjugated to FITC (green) was bound to the cell surface at 4°C, the cells were then further incubated at 37°C for 40 min to allow transport to the Golgi-complex (red). B, Using mask overlay image analysis we quantified the internalization of FITC-ChxB to the GM130-positive Golgi apparatus. Values represent means means ± SE (n>30; **p<0.01, t-test). C, The amount of FITC–ChxB being internalized during a 10-min uptake assay was quantified. FITC-ChxB was bound at 4°C to cell surface; followed by incubation of the cells at 37°C for 10 min to allow internalization. By image analysis we determined the ratio of internalized to total ChxB to obtain a value for the amount of ChxB being internalized. Values represent the means ± SE (n>30 cells, ***p<0.001; t-test).
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Figure S2
Neurons regulate RhoB GTPase activity in Oli-neu cells
Oli-neu cells were transfected with a plasmid encoding for RhoB 3xHA and incubated for ~ 4 and 16 hours with (cnm) or without (ctrl) conditioned neuronal medium before cell lysis. RhoB activity was measured by affinity precipitation of active (GTP-bound) Rho from cell lysates using a GST (glutathione-S-transferase) fusion protein containing the Rho-binding domain of Rhotekin. Precipitated proteins (active) and cell lysates (total) were immunoblotted with monoclonal antibody recognizing the HA tag.
