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. 2016 Aug 22;291(42):22063–22073. doi: 10.1074/jbc.M116.735167

FIGURE 5.

FIGURE 5.

Silencing VAMP3 blocks constitutive NKCC2 exocytic delivery in rat TALs. A, representative Western blot showing masking of surface biotinylation sites by NHS-acetate at 4 °C and reappearance of surface NKCC2 signal after exocytic delivery at 37 °C in TALs transduced with scrambled or VAMP3-shRNA. TALs were treated with vehicle or forskolin + IBMX to stimulate cAMP. The vertical division line separates non-consecutive lanes in the same gel and film. B, quantification of NKCC2 exocytic delivery at 30 min in rat TALs measured as biotinylated NKCC2 at the surface after masking with NHS-acetate. In rats transduced in vivo with VAMP3-shRNA, constitutive NKCC2 exocytic delivery was decreased by 86% after silencing VAMP3. However, cAMP was still able to stimulate NKCC2 exocytic delivery after silencing VAMP3. For every experiment (scrambled-shRNA and VAMP3-shRNA), the difference between the non-NHS-acetate masked fraction and the NHS-acetate masked fraction at time 0 was used as reference to calculate the NHS-acetate masked fraction. The values represent the mean percentages of NHS-acetate masked fraction. Error bars represent ± S.E. *, p < 0.05 versus vehicle; #, p < 0.05 versus vehicle/scrambled-shRNA (n = 6).