Figure 5. .

Analysis of wild-type and R416W GFAP expression in transfected MCF7 cells by immunoblotting. MCF7 cells were transfected with either wild-type (lanes 3 and 4) or R416W GFAP (lanes 5 and 6). Untransfected cells were used as a control (lanes 1 and 2). At 48 h after transfection, cells were collected, lysed with MEB, and centrifuged at 18,000 g for 15 min at 4°C. The resulting supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE, followed by immunoblotting using anti-GFAP (A) and anti-actin (B) antibodies. The blots were developed by the ECL system. Notice that, after transfection into MCF7 cells, both wild-type and mutant GFAP expressed at comparable levels, although proteolyzed GFAP fragments with slightly higher electrophoretic mobilities were also detected. Most of the wild-type GFAP was detected in the pellet fraction (A, lane 4) with a small proportion that remained soluble (A, lane 3), whereas the R416W GFAP was found exclusively in the pellet fraction (A, lane 6). Equal loading of each supernatant and pellet fractions was confirmed by probing with anti-actin antibody (B).