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. Author manuscript; available in PMC: 2010 Sep 4.
Published in final edited form as: Cell. 2009 Sep 4;138(5):990–1004. doi: 10.1016/j.cell.2009.06.047

Figure 3. Knockdown of srGAP2 in cortical neurons reduces axonal and dendritic branching.

Figure 3

(A) Western blot probed with ant-GFP and anti-actin antibodies from COS7 cells co-transfected with either control shRNA plus srGAP2-EGFP (lane 1), srGAP2 shRNA plus srGAP2-EGFP (Dha2, lane 2) or (Dha5, lane3)

(B) Western blot probed with anti-GFP and anti-actin antibodies from COS7 cells co-transfected with either control shRNA plus srGAP2-EGFP (lane 1), srGAP2 shRNA plus srGAP2-EGFP (lane 2), a mutated form of srGAP2*-EGFP (resistant to srGAP2 shRNA) plus control shRNA (lane 3), or srGAP2*-EGFP plus srGAP2 shRNA (lane 4). srGAP2 shRNA significantly knocks down srGAP2 expression compared to control shRNA which can be rescued by expression of srGAP2*-EGFP (compare lanes 3 and 4).

(C–E, G–I) E15 dissociated cortical neurons were cultured for 4 days after ex vivo electroporation with control shRNA, srGAP2 shRNA, or srGAP2 shRNA + srGAP2*-EGFP. Control shRNA transfected neurons display frequent primary branches from the axon (arrowheads in B) and the primary dendrite (arrowheads in F). Both effects were markedly reduced in srGAP2 shRNA transfected neurons (D and H) and rescued by co-transfection of srGAP2 shRNA with srGAP2*-EGFP (E and I).

(E) Quantification of the number of branches from the longest neurite (axon) as shown in C–E.

(I) Quantification of the number of primary dendritic branches as shown in G–I. (Control shRNA, n=42 cells; srGAP2 shRNA, n=95; srGAP2*-EGFP + srGAP2 shRNA, n=39. Cells were taken from 3 independent experiments and analyzed blind to the treatment.

Mann-Whitney Test * p<0.05; ** p<0.01; *** p<0.001.