Figure 4

Phosphorylation of tau by Lck in co-transfected COS-7 cells. DNA constructs coding for V5-tagged tau 2N4R and for Lck were co-transfected into COS-7 cells. In A and B the tyrosine phosphorylation given by wild-type Lck of tau with single replacements of tyrosines is shown, while in C the phosphorylation by activated Lck (LckY505F) of tau with single tyrosines remaining is shown. Lysates were immunoprecipitated with anti-V5 antibody and Western blots were probed with 4G10 antiphosphotyrosine antibody (A top panel, and C) and with anti-tau antibody (A, middle panel), showing approximately equal immunoprecipitation of each tau construct; probing of lysates with anti-Lck antibody (A, bottom panel) showed that approximately equal amounts of Lck were expressed with each tau construct, although Lck alone gave stronger expression. B shows the amounts of immunoprecipitated tau phosphotyrosine after normalisation against immunoprecipitated tau (means of 3 experiments, +/- SEM). WT, wild-type tau (with all 5 tyrosines); YAllF, tau with all five tyrosines replaced by phenylalanines. In A and B, tau with single replacements of a tyrosine with phenylalanine are designated Y18F, Y29F, etc, and in C the mutants with a single tyrosine remaining are designated Y18R, Y29R, etc.