Journal: Acta Neuropathologica

Article Title: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

doi: 10.1007/s00401-016-1564-y

Figure Lengend Snippet: Nefl deletion increases Stat3–stathmin interaction. a Stathmin was immunoprecipitated from sciatic nerve extracts of 34-day-old mice. Western blot shows from left to right, input (Ip) and eluate (E) from wild-type and Nefl − / − mice nerve extracts after anti-IgG control and anti-stathmin pulldown. b Quantification of band intensities in the eluate showed an increased interaction of Stat3 and stathmin in Nefl − / − mice ( t = 17.08; P = 0.0034). Bars represent mean ± SEM (one sample t test, n = 3 independent experiments). c Nefl deletion increases Stat3–stathmin interaction in sciatic nerves from pmn mutant mice. d Western blot analyses show similar levels of stathmin in the sciatic nerve extracts of 34-day-old wild-type, pmn and Nefl − / − mice. Histone levels were determined to ensure equal loading of proteins. e Quantification of Stat3 per stathmin levels in the sciatic nerve extracts (input before immunoprecipitation) from 34-day-old wild-type and Nefl − / − mice ( n = 3 independent experiments). f Stathmin was immunoprecipitated from NSC-34 cell extracts. Western blot shows from left to right input for IgG, mock, and NFL overexpressing NSC-34 cells. Right blot shows eluate after IgG control pulldown and stathmin pulldown from NSC-34 cells after transfection with mock (only lipofectamine) or, NFL overexpression vector, respectively. g Western blot analysis reveals increased levels of phosphorylated Stat3 (at Y705) in the sciatic nerve extracts of 34-day-old pmn and Nefl − / − in comparison to wild-type mice. Quantification of pStat3 normalized by total Stat3 levels in h pmn mouse compared to wild type ( t = 4.928; P = 0.0044; n = 6 independent experiments) and i in Nefl − / − mouse compared to the wild-type mouse ( t = 2.865; P = 0.0242; n = 8 independent experiments). Bars represent mean ± SEM

Article Snippet: Neurofilament-heavy chain antibody (AB5539) was obtained from Millipore, eIF2α (D7D3), p-STAT3 Y705 (D3A7), and Stat3 (124H6) (9139S) antibodies from Cell Signaling Technology. γ-tubulin (clone GTU-88), tau (T-6402), acetylated-α-tubulin (clone 6-11b-1; T7451) and α-tubulin (clone B-5-1-2; T5168) antibodies were purchased from Sigma-Aldrich.

Techniques: Immunoprecipitation, Western Blot, Control, Mutagenesis, Transfection, Over Expression, Plasmid Preparation, Comparison

Journal: Acta Neuropathologica

Article Title: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

doi: 10.1007/s00401-016-1564-y

Figure Lengend Snippet: Distribution of Stat3 and stathmin in axons of wild-type and Nefl − / − motoneurons as revealed by high-resolution SIM. Motoneurons were cultured for 3 days in vitro. Representative images of wild-type and Nefl − / − motoneurons, stained with antibodies against tyrosinated α-tubulin ( green ), Stat3 ( red ), and stathmin ( blue ). The antibody against tyrosinated α-tubulin stains both soluble αβ-tubulin heterodimers and polymerized highly dynamic microtubules. Note that the colocalization of stathmin with Stat3 increases in Nefl − / − motoneurons ( right panel ). Scale bar 20 µm ( first and third lane ). White square boxes indicate the regions enlarged in the second and fourth lane , scale bar 2 µm

Article Snippet: Neurofilament-heavy chain antibody (AB5539) was obtained from Millipore, eIF2α (D7D3), p-STAT3 Y705 (D3A7), and Stat3 (124H6) (9139S) antibodies from Cell Signaling Technology. γ-tubulin (clone GTU-88), tau (T-6402), acetylated-α-tubulin (clone 6-11b-1; T7451) and α-tubulin (clone B-5-1-2; T5168) antibodies were purchased from Sigma-Aldrich.

Techniques: Cell Culture, In Vitro, Staining