Figure 6. Caspase sensitivity of AxD-causing GFAP mutant.
U343MG cells transiently transfected with R239H GFAP were fixed at 48 h after transfection. Cells were processed for double-label immunofluorescence microscopy using SMI-21 anti-GFAP (A and D, green channel) and either D225 (B, red channel) or anti-active caspase 6 (E, red channel) antibodies. Merged image shows the region of colocalization appearing yellow (C and F). Notice that GFAP-rich aggregates in cells transfected with R239H GFAP (A and D, arrows) were also immunopositive for both N-GFAP (B, arrows) and active caspase 6 (E, arrows). Bar, 10 μm. (G) Supernatant (S) and pellet (P) fractions prepared from untransfected (lanes 1 and 3) or R239H GFAP-transfected (lanes 2 and 4) cells were analyzed by immunoblotting with the SMI-21 antibody. Notice a ~26 kDa degradation product was detected in the pellet fraction of R239H-transfected cells (lane 4). (H) The identity of this fragment was confirmed by immunoprecipitating N-GFAP from the pellet fraction (lane 1) with the D225 antibody, followed by immunoblotting with the SMI-21 antibody (lane 2). (I) Immunoblotting analysis of total cell lysates with the SMI-21 antibody showed that cells transfected with R239H GFAP generated N-GFAP (lane 1, p26), whose production was inhibited by the caspase inhibitor zVAD-fmk (lane 2).