Figure 4.

Clathrin-dependent endocytosis of anti-TNFs in dendritic cells. (A) Identification of anti-TNF/TmTNF-associated proteins: Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h and incubated either with isotype matched human IgG1 or with anti-TNF for 5 min at 37°C. Peptide sequences corresponding to TNF or clathrin heavy chain from anti-TNF immunoprecipitated proteins were identified by LC-MS/MS. (B) Identification of anti-TNF/TmTNF-associated clathrin heavy chain: Immunoprecipitated proteins and total cell extracts from DCs as treated in (A) above were subjected to immunoblotting using anti-clathrin heavy chain or anti-TNF antibodies. IP: Immunoprecipitation. (C) Effect of clathrin inhibitor on endocytosis of anti-TNF: Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h either in the absence (Medium) or presence of a 5 μM clathrin inhibitor, Dyngo 4a. Endocytosis of the internalized anti-TNF was measured as an increase in fluorescence intensity by flow cytometry for up to 4 h.