Additional file 2: Figure S1. of Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

A- GST-Tau was incubated with p35/CDK5 or p25/CDK5 and [γ-32P]-ATP for the times indicated, then subjected to SDS-PAGE prior to autoradiography (upper section). Tau bands were digested with Lys-C and phosphopeptides isolated and identified as described in Methods. A major phosphopeptide eluted with mass/charge ratio of 802.4331 corresponding to the peptide containing Ser235 (lower section), and a second doubly phosphorylated peptide corresponding to a peptide including Ser202 and Thr205 was also identified but at much lower abundance. The same result was obtained from two different phosphorylation reactions. B- GST-tau was phosphorylated as above but using non-radioactive ATP. Proteins were transferred to nitrocellulose after SDS-PAGE and probed with the indicated antibodies. Data is representative of two experiments. Figure S2. Co-expression of CDK5 complexes with CRMP2 and CRMP4. Hela cells were co-transfected with equal amounts of expression constructs for CDK5 catalytic subunit, p35 or p25 and FLAG-tagged CRMP2 (A and B) or CRMP4 (C and D) as indicated. Cells were lysed and protein expression and phosphorylation assessed by Western blot analysis (A and C). Quantification was performed on a Licor Odyssey (B and D) with data shown as mean ± S.E.M of three experiments in duplicate. t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Figure S3. Co-expression of CDK5 complexes with tau. Hela cells were co-transfected with equal amounts of expression constructs for CDK5 catalytic subunit, p35 or p25 and tau. Cells were lysed and protein expression and phosphorylation assessed by (A) Western blot analysis. (B-D) Quantification was performed on a Licor Odyssey and the ratio between phospho-tau: total tau calculated for each phospho-tau antibody. Data shown as mean ± S.E.M. for three experiments performed in duplicate. t-test, *P < 0.05, **P < 0.01, ***P < 0.001. (PDF 2557 kb