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Fig. 6. Smad5 activity in cultures of granule cell precursors. The phosphorylation
status of Smad1/5/8 was evaluated by western blotting of cells cultured for 24
hours in the presence of Shh and treated with Bmp2 for different time periods
(0, 5, 15 and 60 minutes). Total cell lysates from granular cell cultures were
separated by SDS-PAGE and the resulting nitrocellulose membranes blotted with
an antibody against phosphorylated-Smad1/5/8 (P-Smad1/5/8) (lysate).
Alternatively, cultures were immunoprecipitated with the anti-Smad5 antibody,
separated by SDS-PAGE and the resulting nitrocellulose membranes blotted with
an antibody against phosphorylated-Smad1/5/8 (P-Smad1/5/8) (IP, Smad5). (B-E)
One-day old granular cell cultures plated on laminin and treated with Shh were
transfected with DNA vectors containing EGFP (B,C) or EGFP-Smad5 (D,E). Cells
were immunostained for proliferation with anti-BrdU (red), with the nuclear
marker DAPI (blue) and with GFP (green). White arrows in B point indicate
transfected cells (green stained in C) that are BrdU labelled. White arrows in
D indicate the position of transfected cells (green stained in F) that are
BrdU negative. (F) The percentage of GFP-expressing cells that were
differentiated cells (cells that do not incorporate BrdU) was evaluated in
each transfection group. Overexpression of Smad5 decreased the percentage of
proliferating cells more than threefold when compared with the control group
transfected with EGFP (from 54.8±8.5% to 21.5±5.3%).
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