Fig. 3. LPS-induced pre-TNF intracellular distribution is not affected by preventing ERK1/2 activation. (A-D) RAW cells were pre-treated for 60 minutes with 10 µM of the TACE inhibitor TAPI-1, with or without 2 µM PD 184352, and then either left untreated or stimulated for 4 hours with 100 ng/ml LPS. The medium was removed, the cells fixed, and thawed cryosections prepared as described in Materials and Methods. The sections were labelled with antibodies against TNF followed by species-specific antibodies and protein-A gold. Micrographs in A-C are taken from the most intensely labelled cells to illustrate the structure of the labelled compartments detected by quantitative methods (D). (A) Labelling is located over lamellipodia-like (Lam) structures and endosome/lysosome profiles (End/Lys); (B) labelling is concentrated in putative peripheral endosomes with pleiomorphic tubulovesicular morphology (arrows); (C) labelling is found over a stack of Golgi cisternae (Golgi). (D) For RAW cells treated with LPS, or PD 184352 plus LPS, values are means of percentage gold counts from two EM grids ± range. PM, plasma membrane; PER, peripherally located tubulo-vesicular structures; DEEP, tubulo-vesicular structures located deep in the cell; MVB/LYS, multi-vesicular bodies; Lys, endolysosomes with heterogeneous content; ER, endoplasmic reticulum; NE, nuclear envelope; MIT, mitochondria. For unbiased counting methods and further details on compartment identification refer to Watt et al. (Watt et al., 2002). Scale bars: 100 nm.