Additional file 2 of Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates

Additional file 2: Figure S1. Intracellular localization of phosphorylated GAP-43 T172 and its responsible kinase. (a) As well as pan-GAP-43 Ab (left), the immunoreactivity of pT172 Ab on full-size membranes (right), showing a wider range of molecular masses, indicated that this Ab mostly recognizes a single band of mouse GAP-43 (mGAP-43) in the total proteins derived from the lysate of cultured mouse cortical neurons (DIV3). (b) In vitro phosphorylation of GAP-43 by activated JNK. Myc-GAP-43 was immunoprecipitaed using anti-myc and used as the JNK substrate. Phosphorylation experiments were performed following the manufacturer’s manual of “JNK1, Acitve” (M33-10g, SignalChem). (c) Immunostaining of cultured hippocampal neurons using pT172 Ab (blue) with rhodamine-phalloidin (red) and Tuj-1 Ab (green). Note that pT172Ab labeled the tubulin-positive area of the axon more intensely than the actin-positive area. Scale bar: 20 μm. (d) pT172Ab immunoreactivity in the growth cone was colocalized with GAP-43 (overlap coefficient: 0.62, calculated by ZEISS ZEN software) at ROI (white box). Scale bar: 10 μm.