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. 2019 Jan 10;14(1):e0210193. doi: 10.1371/journal.pone.0210193

Fig 4. HNK-1 epitope on N1534 of the C domain affects neurite outgrowth.

Fig 4

(A) Scheme of FNIIIx7N1534Q-myc-His (N1534Q). N-glycosylation site (Asn-X-Ser/Thr; highlighted in yellow) at N1534 was mutated to glutamine. (B) N1534Q was purified with or without HNK-1 synthesis enzymes (HNK-1(+) or HNK-1(-), respectively), subjected to SDS-PAGE and stained with CBB or immunoblotted with anti-myc pAb and HNK-1 mAb. Each blot was representative of 2 images of western blots. 1 and 0.2 μg proteins were loaded to each lane for CBB and western blots, respectively. (C) Primary hippocampal neurons were cultured on PDL, PDL coated with N1534Q without HNK-1 expression (HNK-1(-)), or PDL coated with N1534Q with HNK-1 expression (HNK-1(+)). Neurons were immunostained and analyzed at DIV3. Green, PDL; yellow, N1534Q HNK-1(-); and orange, N1534Q HNK-1(+). Numbers of neurons measured on PDL, N1534Q HNK-1(-), and N1534Q HNK-1(+) were 184, 197, 195, respectively. This experiment was performed across two independent cultures, and the representative result obtained from a single culture was shown. Error bars represent SEM. ***p < 0.001; n.s., p > 0.05.