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. 2013 Mar 15;27(6):615–626. doi: 10.1101/gad.212308.112

Figure 7.

Figure 7.

PQBP1-modulated AS of NCAM-140 influences neurite outgrowth. (A) A schematic of alternatively spliced region of NCAM-140. Green rectangles are exons, and black lines are introns. The gray rectangle marks the VASE. Arrowheads are primers designed for RT–PCR and electrophoresis. (B) NCAM-140 VASE splicing isoforms for neuron samples infected with virus from a nontargeting control shRNA or a PQBP1 targeting shRNA were analyzed by RT–PCR and gel electrophoresis. See also Supplemental Figure S6. (C) RT–PCR quantification for VASE− and VASE+. The ratio of the two isoform levels are compared among neurons infected with virus from a nontargeting control shRNA, an unmodified PQBP1 targeting shRNA, or PQBP1 targeting shRNA constructs coexpressing exogenous wild-type (WT) PQBP1, ΔAG, or Y65C. The mean of three independent measurements ± SD is shown. The differences between samples were analyzed by one-way ANOVA test. (**) Statistically significant with P-value < 0.01. (D) Western blot of neurons infected with virus from a nontargeting shRNA, an unmodified PQBP1 targeting shRNA, a PQBP1 targeting shRNA construct coexpressing exogenous wild-type PQBP1, or a PQBP1 targeting shRNA construct coexpressing exogenous VASE− to restore the ratio of VASE−/VASE+. (E) RT–PCR quantification for VASE− and VASE+ expression levels of neurons infected with virus from a nontargeting shRNA, an unmodified PQBP1 targeting shRNA, or a PQBP1 targeting shRNA construct coexpressing exogenous VASE− to restore the ratio of VASE−/VASE+. The mean of three independent measurements ± SD is shown. The differences between samples were analyzed by one-way ANOVA test. (**) Statistically significant with P-value < 0.01. (F) Immunofluorescence of individual neurons that were transiently transfected with a PQBP1 targeting shRNA construct coexpressing exogenous VASE− to restore the ratio of VASE−/VASE+ together with a GFP vector. Neurons were grown on a layer of feeder glial cells. (G) Sholl analysis of dendritic branching for individual neurons that were transiently transfected with a nontargeting control shRNA, an unmodified PQBP1 targeting shRNA, or a PQBP1 targeting shRNA construct coexpressing exogenous VASE− to restore the ratio of VASE−/VASE+ (mean ± SE, n = 15–17).