They were tagged with EGFP at the amino terminus. pEGFP OPTN2 bp AG insertion and pEGFP OPTNQ398X were made by site directed mutagenesis based on pEGFP OPTNWT. Constructs pEGFP OPTN217 398 selleck bio and pEGFP Inhibitors,Modulators,Libraries OPTN217 577 were made by digestion of plasmids pEGFP OPTNQ398X and pEGFP OPTNWT with BglII, gel purification and self ligation. All the constructs were verified by sequence analyses. Immunofluorscence staining RGC5 or Neuro2A cells plated on Lab Tek 8 well CC2 glass chamber slides were transfected with Lipofectamine LTX transfection reagent for 18 hours and fixed with 4% paraformaldehyde for 15 min. After permeabilization in 0. 2% Triton X 100 for 4 min, the cells were blocked for 1 hour with 3% bovine serum albumin and incubated at room temperature for 1 hour with mouse monoclonal anti GM130 pri mary antibody.

After a further 1 hour incubation with Cy3 goat anti mouse IgG, the slides were mounted in Vectashield mounting solution containing 4,6 diamidino 2 phenylindole. The fluorescence was visualized on an Axioscope with a 63�� oil objective. Approximately 20 images were acquired for each Inhibitors,Modulators,Libraries specimen. The images were evaluated for formation of foci which are bright, granular or punctate structures located in the perinuclear region of the cell. Additionally, the percentage of cells with fragmented Golgi was determined. Golgi fragmentation was defined as the appearance of disconnected, small and round Golgi fragments dispersed in the cells. Cells with compromised Golgi were counted and the percentage of cells containing fragmented Golgi relative to the total number of transfected cells was cal culated.

A minimum of 40 cells were evaluated for each expression vector per experiment with the exception of D474N and 2 bp AG insertion constructs with Inhibitors,Modulators,Libraries which only approximately 20 cells were examined. Four independent ex periments were performed. Results were presented as the average of the 4 experiments. Transferrin Inhibitors,Modulators,Libraries uptake RGC5 cells plated on Lab Tek 8 well CC2 glass chamber Inhibitors,Modulators,Libraries slides were transfected for 18 hours. Thereafter, the cells were washed with phosphate buffered saline and incubated for 1 hour in serum free DMEM with 0. 2% BSA to deplete serum. The cells were then incubated with DMEM BSA containing 25 ug ml of Texas red transferrin at 37 C for 0 or 15 min, placed on ice, and washed 3 times with cold PBS containing 0. 2% BSA, 1 mM CaCl2 and 1 mM MgCl2.

Following a wash in cold acid buffer containing 0. 2 M acetic acid and 0. 5 M NaCl and a rinse with ice cold PBS, the cells were fixed in 4% paraformaldehyde and mounted. Images were acquired with Leica SP2 confocal system with a 40�� dry ob jective using sequential scanning to check details minimize the bleed through. The uptake of TR Tf was quantified as described by Park et al. In brief, the outline of single cell was drawn and using Leica confocal software, the average fluorescence intensity of TR Tf inside the cell was mea sured.