Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling

In stimulated neutrophils, the majority of tyrosine-phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells that are lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserve and solubilize tyrosine-phosphorylated proteins but also retain their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilization of a significant fraction of tyrosine-phosphorylated proteins. Furthermore, we observed in neutrophils in which CD32 was cross-linked that the tyrosine kinase activity of Lyn was enhanced in the soluble fraction recovered from the hypertonic lysis but not in that derived from the first hypotonic lysis. Furthermore, we detected tyrosine kinase activity and the presence of the tyrosine kinase Syk in association with CD32 in the soluble hypertonic lysis fraction. This fraction also contained most of the tyrosine-phosphorylated proteins including Cbl, Syk and CD32 itself. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.