Demonstration of the presence of p35Nck5a in cells with monocytic phenotype.
(A) Presence of a 35-kd protein immunoreactive with an antibody to Nck5a in HL60 cells induced to monocyte phenotype by exposure to 10−7M 1,25D3, or to macrophage phenotype by 10−8M TPA for 96 hours. The migration of size markers is indicated on the left side of the panel in kilodaltons. (B) An immunoblot (IB) run in parallel to the one shown in panel A, but probed with antibody that was preblocked with the immunizing peptide. Note the absence of the 35-kd band. (C) The 3 upper panels show IBs for the indicated proteins (Cal indicates calreticulin, a loading control) in lysates of monocytes and lymphocytes from blood of healthy volunteers, and from undifferentiated (negative control) and differentiated HL60 cells (10−7M 1,25D3 for 96 hours) as a positive control. The fourth panel shows kinase activity associated with Cdk5 immunoprecipitated (IP) from these lysates using histone H1 (H1) as the substrate and the bottom panel the Cdk5 protein content in these IPs demonstrated by immunoblotting. (D) Similar analysis of lymphocytic cell lines. (E) Absence of p35Nck5a in HL60 induced toward granulocytic phenotype by 96 hours of treatment with 10−6M 9-cis retinoic acid (cisRA) or all-trans retinoic acid (atRA). HL60 cells treated with monocyte/macrophage inducers show strong p35 bands, whereas cells treated with 1.25% DMSO for 96 hours show a faint 35-kd band. The bottom panels demonstrate Cdk5- and p35Nck5a-associated kinase activity showing a strong signal for 1,25D3-treated HL60 cells, a weak signal for DMSO-treated cells, and only background levels in untreated RA-treated or TPA-treated cells. The background level was determined by incubation of the extract with beads that were not coated with the antibody to either Cdk5 or p35Nck5a (marked NS, nonspecific). The data shown illustrate 4 similar experiments.
