Fig. 6
MAP1B was identified as a target of DOK6 by GST pull-down and mass spectrometry. a The E18.5 mouse purified brain tissue protein was incubated with the DOK6-GST fusion protein, the binding protein was retained, and then the bands were silver-stained, the red arrow indicates the location of the DOK6-GST fusion protein. Mass spectrometry analysis was performed on Lane 3, Lane 4, and Lane 5. Lane 3 and Lane 5 proteins were employed as controls to identify differential proteins. b The process of selecting possible interacting proteins by phosphoproteomic and GST pulldown assays. Phosphotomics mass spectrometry and GST pulldown mass spectrometry were analyzed and classified to screen candidate proteins interacting with DOK6. DOK6 potential interacting proteins, the fold change, and score were obtained based on mass spectrometry results and are listed in the lower panel. c The top ten GO pathways were analyzed with data from phosphoproteomics mass spectrometry assays on pulldown samples. d Coimmunoprecipitation assay of DOK6 with lysates from P3 mice sciatic nerve tissue. Precipitates and total lysates were subjected to Western blotting with antibodies against TrkA, TrkB, TrkC, RET, p75NTR, and MAP1B, revealing that DOK6 was able to bind to MAP1B. e–j Protein levels of P3 sciatic nerve protein associated with the MEK/ERK, p38, PI3K/AKT, PLCγ, JNK and MEK5/ERK5 signaling pathway assessed by Western blot in the control and Dok6−/− groups. k Schematic diagram of the MEK/ERK signaling pathway in which DOK6 is involved. Data are presented as the mean ± SD, ***p < 0.001, two-tailed unpaired Student’s t test in Control and mutant mice
