Figure 2.

Effect of C(1-3)A and C(11-14)A substitutions on aggregation of CSPα ANCL mutants. PC12 cells were transfected for 48 hours with EGFP-tagged or untagged CSPα constructs. Cell lysates were resolved by SDS-PAGE and analysed by immunoblotting with anti-GFP antibody (A and D) and anti-CSPα antibody (C and F). a indicates aggregates, p shows position of palmitoylated monomeric CSPα, and np designates the non-palmitoylated monomers. Position of size markers are shown on the left hand side. Asterisks in panels C and F indicate endogenous palmitoylated CSPα. In panel C, the immunoblot on the right provides a comparison of CSPα expression levels in untransfected and transfected cells. Quantification of EGFP-CSPα proteins is presented as a ratio of aggregated to monomeric + dimeric forms of the proteins (n = 4), shown together with SEM (error bars) (panels B and E). The data was analysed using an unpaired Student’s T test; asterisks denote a significant difference (*p < 0.05, **p < 0.01) from the respective control CSPα construct (wild-type, ΔL116 or L115R).