Figure 4

PTN-induced CDK5 activation depends on RPTPβ/ζ but not α_νβ3 integrin. (a) CDK5 activity was measured using the ADP-Glo Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). (b) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. (c) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α_νβ₃ interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). (d) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β₃Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β₃Tyr773 (pβ₃) and total β₃ (tβ₃) amounts were quantified and the ratio pβ₃/tβ₃ was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β₃Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for (a), 7.8 for (b), 18.2 for (c) and 11.9 for (d).