Figure 5

Subcellular tyrosine-phosphorylation pattern of VCP/p97. A. Immunoprecipitation of subcellular compartments during U937 differentiation and subsequent Western blot analysis. Cytosol, membranes, and nuclei of undifferentiated U937 control cells (U937) and 11d differentiated populations (TPA-differentiated U937), respectively, were immunoprecipitated with an anti-phosphotyrosine antibody (IP: P-tyr) followed by an anti-VCP/p97 Western blot (WB: VCP). A cross-immunoprecipitation was performed with anti-VCP/p97 (IP: VCP) and subsequent anti-phosphotyrosine Western blot (WB: P-tyr). Protein controls were evaluated by anti-VCP/p97 immunoprecipitation (IP: VCP) followed by anti-VCP/p97 Western blot (WB: VCP). B. 2D-Western blot analysis of cytosolic and nuclear fractions during U937 differentiation. Cytosolic and nuclear proteins from U937 control cells (U937) and 7d TPA-differentiated cells were separated by non-linear isoelectric focusing (pH 3–10, NL) and subsequent 10% SDS-PAGE in the second dimension. Western blot analysis was performed with an anti-VCP/p97 antibody (VCP) and after successful stripping, which revealed no detectable spots anymore (data not shown), the same blots were incubated with an anti-phosphotyrosine antibody (P-tyr) and developed by the ECL kit.