Fig. 1. Effect of protein kinase inhibitors on TLR-induced production of intracellular and extracellular TNF in murine macrophages. (A) RAW cells were pre-treated for 60 minutes with or without 2 µM PD 184352 (PD) and then either left untreated or were stimulated for 4 hours with 100 ng/ml LPS. The cell culture supernatants were collected and the levels of TNF analysed using the beadlyte system (histogram). The cell lysates (30 µg protein) were subjected to SDS-PAGE and, after transfer to nitrocellulose, the membrane was probed with a murine anti-TNF-specific antibody or an anti-actin antibody (lower panel). (B) As in A, except that BMDM were used instead of RAW cells. (C) As in A, except that primary human monocytes-macrophages were used instead of RAW cells and 10 µM U0126 (U0) was used as an additional MKK1 inhibitor. (D) As in A, except that cells were stimulated for 4 hours with 100 ng/ml FSL1. The results are shown as mean±s.e.m. for three (A,B) or two (C,D) separate experiments.