Figure 2. Agnoprotein is released by glial cells.

A. Immunoprecipitation of agnoprotein in growth media. T98G cells were transfected with pSELECT-GFPzeo control plasmid encoding GFP or with pSELECT-GFPzeo-agno expression plasmid which contained two transcription units, the first drove the expression of agnoprotein and the second drove the expression of GFP. Growth medium was collected at 48h post-transfections simultaneously along with the whole cell protein extracts. Agnoprotein was immunoprecipitated in the growth medium, and analyzed by western blotting (lanes 6 – 10). Whole cell protein extracts were also processed and loaded in parallel for the cellular expression of agnoprotein (lanes 1 – 5). Ab-Lc points the light chain of agnoprotein antibody. B. Immunoprecipitation of GFP in growth medium of T98G cells. GFP was immunoprecipitated in the growth media from the same set of experiments presented in panel A and analyzed by western blot (lanes 6 – 10). Whole cell protein extracts were also processed and loaded in parallel for the cellular expression of GFP (lanes 1 – 5). Efficiency of anti-GFP antibody for immunoprecipitating GFP protein was also assessed in whole cell protein lysates and shown (right panel, lanes 7–9). Ab-Hc indicates the IgG heavy chain of the antibody. C. T98G cells were transfected with expression plasmids encoding either GFP alone or GFP and agnoprotein together as described above. Live-fluorescein images of GFP expression was taken at 48 hrs posttransfections. Transfection efficiencies were determined as ~40% for GFP control and ~43% for GFP + agnoprotein by calculating the percentile of GFP positive cells over total number of cells. D. T98G cells were transfected with expression plasmids encoding either GFP alone or GFP and agnoprotein together as described in panel A and B. Forty eight hours post transfections, MTT activities were detected. MTT activity of untransfected cells is presented as 100%, and MTT activity of the transfected groups is presented proportionally.