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. 2013 Sep 3;288(41):29453–29466. doi: 10.1074/jbc.M113.468801

FIGURE 9.

FIGURE 9.

Knockdown of endogenous SNX26 by shRNA causes alterations in dendritic spine formation, which can be partially rescued by resistant mouse SNX26. A, HEK293T cells were co-transfected with GFP-mSNX26 or GFP-mSNX26-res and shRNA-scr or shRNA-SNX26, respectively. The knockdown efficiency of shRNA-SNX26 was confirmed by immunostaining or Western blotting with anti-GFP antibody. Although the expression of GFP-mSNX26 was successfully reduced by shRNA-SNX26, that of GFP-mSNX26-res was not affected by shRNA-SNX26. The term, res, indicates silent mutation that is resistant to shRNA-SNX26. Scale bar, 10 μm; βTub, β-tubulin. B, endogenous expression of SNX26 in cultured hippocampal neurons was reduced by shRNA-SNX26. Hippocampal neurons were transfected with shRNA-scr or shRNA-SNX26 at DIV 8, fixed at DIV 13, and immunostained with anti-SNX26 antibody, followed by Alexa 488-conjugated donkey anti-goat antibody. Alexa 488 channel shows endogenous levels of SNX26. dsRed channel shows the neurons transfected with shRNA-scr or shRNA-SNX26. Arrowheads indicated the soma of neurons transfected with shRNAs. The neuron expressing shRNA-SNX26 showed the reduced levels of SNX26 expression at soma compared with nontransfected neurons (arrows). Scale bar, 10 μm. C–H, cultured hippocampal neurons were transfected at DIV 8 with shRNA-scr or shRNA-SNX26 with GFP or shRNA-SNX26 with GFP-mSNX26-res and fixed at DIV 13. Quantification of the number of total protrusions, spines, and filopodia with expression of each group is shown. Spines were defined as dendritic protrusions of <5 μm in length with heads and filopodia as dendritic protrusions of <10 μm in length without heads. Data were collected from 22 to 42 dendrites of 12–24 neurons for each group in three independent experiments. *, p < 0.01 (ANOVA and Tukey's HDS post hoc test); ***, p < 0.001 (Student's t test). G, representative images of spine morphologies from the dendrite in the neuron expressing shRNA-scr or shRNA-SNX26 with GFP or shRNA-SNX26 with GFP-mSNX26-res, respectively. Scale bar, 15 μm. G, pie graphs illustrating the changes in the portion of different morphological types of spine protrusions in the neurons expressing shRNA-scr or shRNA-SNX26 with GFP or shRNA-SNX26 with GFP-mSNX26-res.