Figure 9. .

The similarity between Rosenthal fibers and GFAP aggregates formed in transfected human astrocytoma cells. U343MG cells were transiently transfected with R416W GFAP and were routinely stained with rabbit polyclonal antibodies (3270) to GFAP (A, C, E, and G) and then were double stained with mouse monoclonal antibodies specific to R416W GFAP (B), αB-crystallin (αB-cry) (D), or HSP27 (F). Notice that the GFAP containing aggregates are also positive for both αB-crystallin and HSP27 (arrows in C–F). To demonstrate the presence of ubiquitin in the GFAP aggregates, cells were cotransfected with His₆-myc ubiquitin as well as R416W GFAP and then were stained with rabbit polyclonal antibodies to GFAP (G) and the mouse monoclonal antibodies that recognize the myc epitope (H), showing that the GFAP aggregates contain ubiquitin. Bars = 10 μm. I, Wild-type and R416W GFAP (R416W) were transiently expressed in the human astrocyte cell line U343MG, and supernatant (S) and pellet (P) fractions were prepared from these culture and were compared with mock transfected cells. Cell fractionation used HEB, which almost completely solubilized wild-type GFAP. R416W GFAP, on the other hand, remained in the pellet fraction. Immunoblots of the cell fractions were probed with antibodies to GFAP, αB-crystallin, HSP27, HSP70, and finally actin, which was used as a loading control. Notice that, when cells were transfected with R416W GFAP, a significant proportion of the HSP27 and αB-crystallin but not HSP70 remained in the pellet fraction along with the R416W GFAP. Both the sHSPs and R416W GFAP were more resistant to extraction compared with these proteins in the wild-type GFAP transfected cells.