Fig. 2

Specificity of novel pSer129 αS antibodies as determined by immunoblotting with NFL and αS proteins phosphorylated in vitro with PLK3 or CKII. NFL, S473A NFL, αS and S129A αS were incubated with PLK3 or CKII. The proteins were resolved onto 13 % polyacrylamide gels and analyzed by immunoblotting with novel αS antibodies (as indicated above each blot) LS7, LS11, LC2-2C2, LS4-1B1, LS4-2C3 and LS4-2G12 and novel NFL antibody 4F8, to demonstrate their specificity. NR4 (anti-NFL) and Syn 204 (anti-αS) were included to show each protein, respectively. Previously reported pSer129 αS antibodies 81A and EP1536Y were included for comparison. Reactions were performed in duplicate. Antibody 4F8, generated against the pSer473 NFL epitope, can cross react with αS phosphorylated at Ser129. Antibodies 81A, LS11, LS4-1B1 and LS4-2C3 generated against the pSer129 αS epitope can cross react with NFL phosphorylated at Ser473. Antibodies EP1536Y, LS3-2C2 and LS4-2G12 generated against the pSer129 αS epitope reacted only with phosphorylated WT αS. Antibody LS7 generated against the pSer129 αS epitope detected both phospho- and non-phospho-αS. The mobility of molecular mass markers are shown on the left