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. 2017 Jan 31;7:10. doi: 10.1038/s41598-017-00036-8

Figure 4.

Figure 4

C(4-7)L substitutions block aggregation of the ANCL mutants. EGFP-CSPα wild-type and ΔL116/L115R constructs with or without C(4-7)L substitutions were transfected into PC12 cells for 48 hours and subsequently analysed by immunoblotting with anti-GFP (A). The non-palmitoylated monomers (np), palmitoylated monomers (p) and aggregates (a) are marked by arrows. The position of molecular marker is shown on the left hand side. Averaged data of the aggregate to monomer + dimer ratio (n = 4) is shown together with SEM (B). Statistical significance was assessed using an unpaired Student T test, asterisks denote a significant difference compared with the control (wild-type, ΔL116 or L115R) CSPα construct (***p < 0.001). (C) PC12 cells transfected with the wild-type and mutant EGFP-CSPα constructs with or without the C(4-7)L substitutions were treated with 30 µg/ml of BFA for 4 hours. The samples were then resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using an antibody against GFP. The untreated and BFA-treated samples that are shown are from the same immunoblot but with intervening gel lanes removed and this is indicated by the solid black line.