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. 2013 Dec 15;24(24):3857–3868. doi: 10.1091/mbc.E13-06-0333

FIGURE 5:

FIGURE 5:

Bcr regulates polarity by restricting PKCζ function. (A) PKCζ inhibitor can partially rescue protrusion formation in Bcr−/− cortical mouse astrocytes. Representative images showing WT and Bcr−/− astrocytes treated overnight with PBS (control) or 10 μM of PKCζ pseudosubstrate inhibitor and then fixed and immunostained for acetylated tubulin. Dashed white line represents the scratch. Scale bar: 10 μm. n = ∼100; N = 3. (B) PKCζ inhibitor can partially rescue centrosome reorientation in Bcr−/− cortical mouse astrocytes. Quantification of polarized centrosomes in WT and Bcr−/− cortical mouse astrocytes treated overnight with PBS (control) or 10 μM of PKCζ pseudosubstrate inhibitor. n = ∼100; N = 3. (C) Overexpression of Bcr, but not Abr, negatively regulates PKCζ levels. Western blot analysis was performed on lysates from COS7 cells expressing control (empty vector) or myc-tagged Tiam1, Bcr, or Abr. Lysates were immunoblotted with α-PKCζ antibodies to assess PKCζ levels and α-GAPDH for a loading control. N = 3. (D) Quantification of PKCζ levels from (C). N = 3. (E) Bcr and Abr overexpression reduces Pak phosphorylation in COS7 cells. Western blot analysis of lysates from COS7 cells expressing control (myc) or myc-tagged Tiam1, Tiam1 and Bcr, or Tiam1 and Abr, immunoblotted for PKCζ. Lysates were blotted with an α-GAPDH antibody for a loading control. (F) Quantification of PKCζ levels from (E). N = 3. (G) Expression of Bcr, but not Abr, rescues protrusion defects in Bcr−/− cortical mouse astrocytes. WT astrocytes were transfected with eGFP as a positive control. Bcr−/− astrocytes were transfected with eGFP alone or in combination with Bcr or Abr expression plasmids, and then astrocytes were subjected to scratch assays. Cells were then fixed 18 h postscratch and immunostained for acetylated tubulin (red). Yellow dashed line represents scratch. Scale bar: 10 μm. (H) Quantification of the protrusion assay. n = ∼100; N = 3. (I) Expression of Bcr, but not Abr, can rescue centrosome reorientation defects in Bcr−/− cortical mouse astrocytes. Quantification of polarized centrosomes in WT astrocytes transfected with eGFP (positive control) or Bcr−/− cortical mouse astrocytes transfected with eGFP, eGFP and Bcr, or eGFP and Abr. n = ∼100; N = 3. Data are shown ± SEM.