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. 2012 Oct 29;199(3):437–451. doi: 10.1083/jcb.201203109

Figure 4.

Figure 4.

Stathmin knockdown rescues axonal pathology in pmn mutant motoneurons. (A) Colocalization of STAT3-EYFP and stathmin in cultured motoneurons. Both proteins are colocalized and enriched in branch points (arrowheads) and growth cones (arrows). Bars, 10 µm. (B) Immunoprecipitation of stathmin from motoneurons cultured for 5 DIV. Western blot analysis after immunoprecipitation shows that STAT3 interaction with stathmin is enhanced after CNTF application. (first and second lanes) Input (In) and eluate (E) from IgG control; (third and fourth lanes) input and eluate from motoneurons cultured with BDNF; (fifth and sixth lanes) input and eluate from motoneurons cultured with BDNF and pulsed with CNTF for 30 min on day 5. (C) Quantification of Western blot signals shows a twofold increase in the STAT3–stathmin interaction and reduced stathmin–tyrosinated (Tyr) tubulin interaction after CNTF application; n = 3 independent experiments. (D) Immunoprecipitation experiments of stathmin with wild-type and dominant-negative STAT3 (STAT3Y705F-EYFP). Subsequent Western blot analysis shows loss of STAT3–stathmin interaction when phosphorylation at Y705 is abolished. White line indicates that intervening lanes have been spliced out. (E) Western blot analysis of protein extracts from cultured primary motoneurons after lentiviral stathmin knockdown. (F) Axon length is restored in pmn mutant motoneurons after lentiviral stathmin knockdown. Wild-type and pmn mutant motoneurons were cultured for 5 DIV. Stathmin knockdown rescues axon length in pmn mutant motoneurons. CNTF application did not induce additional axon growth in motoneurons with stathmin knockdown. Numbers in bars indicate number of cells measured. n = 3 independent experiments. ***, P < 0.001; Kruskal-Wallis with Dunn’s multiple comparison test. (G) Representative images of pmn mutant motoneurons after lentiviral stathmin knockdown. Cells were labeled with GFP and α-tubulin (Cy-3) antibodies. Bars, 100 µm. (H) CNTF application does not alter stathmin protein level in primary motoneurons of wild-type and pmn mutant mice. BDNF and CNTF are indicated by B and C, respectively. IP, immunoprecipitation; wt, wild type. Error bars represent means ± SEM.