Figure 1.

Scramblase activity is responsible for PS exposure on degenerating axons. (A) The shRNA-mediated downregulation of abc1, tmem16f, or xkr8 expression was performed in cultured DRG neurons using lentivirus vectors. The downregulated expression of abc1, tmem16f, or xkr8 by 2 independent shRNAs used in this study was confirmed by quantitative RT-PCR. The expression levels of each molecule normalized to β-actin are shown relative to that of the control (mean ± SEM, n = 5). (B, C) PS exposure on the transected axons was monitored using the specific probe Alexa594-labeled Annexin V (red). DTAF (green) was used to stain axons. Representative photomicrographs of the fluorescent signal in transected axons expressing the indicated molecules are shown in B. Scale bar = 25 μm. Quantified levels of fluorescent intensities for Annexin V normalized to DTAF relative to the level in the control (labeled as “no infection“) are shown in (C)(mean ± SEM, n = 5). Note that PS exposure is reduced on axons expressing shRNAs for abc1 or xkr8, but not tmem16f. Significant differences from the control (*, P < 0.05; **, P < 0.01) were determined by a one-way ANOVA with Tukey's post-hoc test.