title: Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling.
abstract: 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.
authors.affiliation: Centre de Recherche en Rhumatologie et Immunologie, CIHR group on the Molecular Mechanisms of Inflammation, Centre de Recherche du CHUL, Laval University, Ste.-Foy, Québec, Canada.
authors.forename: Caroline
authors.initials: C
authors.lastname: Gilbert
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Rollet-Labelle
authors.forename: Adriana C
authors.initials: AC
authors.lastname: Caon
authors.forename: Paul H
authors.initials: PH
authors.lastname: Naccache
mesh_terms: D015153:Blotting, Western; D002021:Buffers; D003902:Detergents; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009504:Neutrophils / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D012995:Solubility; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D002021:Buffers; D003902:Detergents; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D017452:Receptors, IgG; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases