Figure 5.

AnkG, myelin and voltage-gated sodium channels (panNaV) protein expression during AIS development in visual cortex. (A) Myelin distribution in layers II/III in an adult mouse (P67) with ankG (green) and myelin basic protein (MBP, red). MBP immunosignals were only rarely observed (arrows). (B) Myelin distribution in layers V and VI in the same section as (A). MBP is more evident in infragranular than supragranular layers. (C) Representative immunoblot bands for ankG and α/β tubulin in total cortex samples from P7 and P15, and upper layer lysates from P21 through >P180 ages. (D) Quantification of ankG expression at various time points during development. P7 and P15 bars represent total visual cortex lysates, ages P21 and older the upper layer fractions only. AnkG protein was normalized to the internal loading control (α/β Tubulin) and levels are expressed relative to the normalized ankG expression at P7, which was set to 1. Shown are bars with standard deviation from three replicates. AnkG expression increased significantly during postnatal development (P7–21). From P21 to 28, coinciding with the maximum of length reduction (Figure 3A), ankG protein was reduced. However, despite significant re-elongation of the AIS past P28 (Figure 3A), no significant changes in protein amount were detected (p ≥ 0.05). (E) Representative immunoblot bands for ankG, MBP, and α/β tubulin in various age samples from P21 through >P180 were separated for upper and lower cortical layers (UL, upper layers; LL, lower layers). P7 and P15 samples were obtained from whole visual cortex lysates. MBP first appeared around P21. Far right lane shows immunoblot from a cerebellar ankG knockout mouse confirming specificity of rabbit anti ankG antibody. (F) PanNa_V immunoblot from the three stages of the tri-phasic AIS length development shows constant levels for sodium channel expression despite significant AIS length changes (Figure 3A). Scale bars (A,B) = 25 μm.