14-3-3 affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

JCS03219 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Absence of 14-3-3η in GFAP antibody immunoprecipitated complex in astrocytes. The astrocyte extracts (lane 1) were subjected to immunoprecipitation with a rabbit preimmune IgG (lane 2) or a rabbit anti-GFAP antibody (lane 3) and subsequent western blot analysis with goat anti-14-3-3η polyclonal antibody (top) or mouse anti-14-3-3γ antibody (bottom). The pellet was also detected by 14-3-3η and 14-3-3γ antibodies (lane 4).

• Supplemental Figure 2 -

  Fig. S2. Vimentin immunoprecipitation from SW13 Cl1 cells by hyperphosphorylation and dephosphorylation treatments. The cells were treated with serum-deprived medium (lanes 2 and 4) or medium containing 100 nM calyculin A (lanes 1 and 3). Then the cell lysate was incubated with buffer alone (lanes 1 and 2) or with CIAP (lanes 3 and 4). The immunoprecipitates were blotted with anti-vimentin or anti-14-3-3γ antibody.

• Supplemental Figure 3 -

  Fig. S3. 14-3-3γ-specific siRNA had no effect on the phenotype of glial filaments in C6 cells. (A) Western blot analysis of 14-3-3γ, actin and GFAP in cell lysates derived from cells untransfected (lane −) or transfected with 14-3-3γ siRNA vector (lane +).(B) Distribution of glial filaments in the population of cells untransfected (control), transfected with pSilencer 2.1-U6 neo vector (vector) or 14-3-3γ siRNA vector (RNAi). A minimum of 100 cells was randomly scored in a blinded manner. Each transfection was repeated three times with similar results.