Unaltered Slack expression in WT mice after peripheral nerve injury. (A,B) Double-labeling immunostaining of Slack and the neuronal marker anti-βIII-tubulin (TUBB3) in DRGs of naive WT mice and 14 days after SNI. Colocalization of Slack and TUBB3 appears in yellow. The staining suggests that Slack is exclusively localized to neurons and that its distribution is not altered in response to the injury. The absence of Slack immunoreactivity in DRGs of Slack-/- mice confirms the antibody specificity (A). The percentage of Slack-immunoreactive DRG neurons of all βIII-tubulin-stained neurons is similar in naive mice and 14 days after SNI ((B); 1833 and 1630 cells counted, respectively; n = 3 mice per group). Student’s t-test: p = 0.9130. (C), Quantitative RT-PCR experiments showed that Slack mRNA levels are not altered in DRGs 7 or 14 days after SNI as compared to naive control animals (n = 8 mice per group). One-way ANOVA: p = 0.1433. (D), Immunostaining in the lumbar spinal cord of WT mice 14 d after SNI shows similar Slack expression in the ipsilateral and contralateral dorsal horn. The absence of Slack immunoreactivity in the spinal cord of Slack-/- mice confirms the antibody specificity. (E,F) A Western blot of spinal cord extracts shows similar Slack protein (140 kDa) expression in naive mice and 7 or 14 days after SNI surgery (E). Uncropped original image is shown in Figure S2A. Quantification is shown in (F). Alpha-tubulin was used as a loading control. One-way ANOVA: p = 0.4446. Bars denote mean ± SEM. Scale bars: 50 µm (A), 200 µm (D).