Figure 5.
Inhibition by soluble laminin 5 (lam5) of early kinases phosphorylation events activated via the CD3–TCR complex. Jurkat cells at 20 × 106/ml in RPMI 5% FCS were incubated with Abs at 37°C for 2.5 min, solubilized, and lysates were immunoprecipitated with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a pAb anti-fyn. After SDS- PAGE separation and electrotransfer onto Immobilon P membrane, proteins were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For densitometric analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned under each lane. For reprobing, the membranes were submerged in stripping buffer, blocked and immuno-detected with (a) pAb anti–Zap 70, (b) pAb anti-lck, and (c) pAb anti-fyn was performed. Reactions were revealed by incubating membranes with GAR HRP followed by ECL. Laminin 5 (lam5) was added in soluble form at 1 μg/ml for Zap 70 and fyn analysis and 5 μg/ml for lck analysis.