FIGURE 5.

PKCδ is required for MerTK cleavage. A, primary macrophages were pretreated for 30 min with 250 nm Go6976 or 250 nm Go6983 in complete medium and subsequently treated with 50 ng/ml LPS. Subsequently, levels of sMER from cell supernatants and levels of full-length (FULL) MERTK from cell extracts were measured by Western blot. B, primary macrophages were incubated with PKCδ siRNA for 48 h, and the top panel exhibits representative knockdown efficiency of two PKCδ siRNAs (δ1 and δ2) by Western blot. In parallel, macrophages were cultured with LPS in the presence of PKCδ siRNA and scrambled (sc) control and sMER measured from supernatants and full-length MERTK from cell extracts by immunoblot. Densitometric analysis is shown to the right after knockdown with both PKCδ siRNAs. C, membrane translocation of PKCδ and phospho-PKCδ (PKCδ-P) post-LPS was determined by immunoblot after isolation of membrane pellets as described under “Materials and Methods.” Membrane translocation was also measured after treatment with NAC (right). M, membranous fraction; C, cytosolic fraction; T, total cellular lysate. Error bars, S.E.