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. 2002 Mar 4;195(5):559–570. doi: 10.1084/jem.20011788

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Copyright © 2002, The Rockefeller University Press

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Figure 4. — TLR4 staining in m-IC_cl2 cells is localized to the Golgi apparatus. (A) Double immunofluorescence of m-IC_cl2 cells for TLR4 and p58K, β-COP, or CTR433, which are all three marker proteins of the Golgi apparatus. (B) Immunofluorescence for TLR4 in m-IC_cl2 cells after pharmacological interference with the structural integrity of the Golgi apparatus. Cells were left untreated or preincubated with 10 μM nocodazole, or 5 μg/ml brefeldin A for the indicated time period before fixation. Note that perinuclear TLR4 staining begins to be concentrated again after 60 min because of the reversible effect and short half life of brefeldin A. (C) TLR4 is permanently located in the Golgi apparatus. Immunostaining of TLR4 in m-IC_cl2 cells after the inhibition of protein synthesis with cycloheximide (50 μg/ml) for 1.5 and 3 h before fixation. As a control, cells were stained with antibodies directed against cyclin B1, a protein with a short half-life. (D) TLR4 is not involved in retrograde membrane trans-Golgi network traffic. The right part of the figure shows cells incubated with the primary antibody for 2 h on ice for chase experiments. After washing with ice-cold PBS, cells were incubated at 37°C for 60 min, fixed, and stained with the secondary fluorescence-labeled antibody. To confirm cell viability, biotin-labeled anti-MHC class I (H-2K^b) antibodies and TxR-labeled streptavidin were used during primary incubation, inducing cross-linking and internalization of labeled MHC class I molecules. The left part of the figure demonstrates complete staining of acetone-fixed cells as a control. ×1,000. (E) Deglycosylation of m-IC_cl2 cells in situ. Before staining with TLR4 or Con A, cells were treated with Endo H or PNGase F. ×1,000.

Figure 4. — TLR4 staining in m-IC_cl2 cells is localized to the Golgi apparatus. (A) Double immunofluorescence of m-IC_cl2 cells for TLR4 and p58K, β-COP, or CTR433, which are all three marker proteins of the Golgi apparatus. (B) Immunofluorescence for TLR4 in m-IC_cl2 cells after pharmacological interference with the structural integrity of the Golgi apparatus. Cells were left untreated or preincubated with 10 μM nocodazole, or 5 μg/ml brefeldin A for the indicated time period before fixation. Note that perinuclear TLR4 staining begins to be concentrated again after 60 min because of the reversible effect and short half life of brefeldin A. (C) TLR4 is permanently located in the Golgi apparatus. Immunostaining of TLR4 in m-IC_cl2 cells after the inhibition of protein synthesis with cycloheximide (50 μg/ml) for 1.5 and 3 h before fixation. As a control, cells were stained with antibodies directed against cyclin B1, a protein with a short half-life. (D) TLR4 is not involved in retrograde membrane trans-Golgi network traffic. The right part of the figure shows cells incubated with the primary antibody for 2 h on ice for chase experiments. After washing with ice-cold PBS, cells were incubated at 37°C for 60 min, fixed, and stained with the secondary fluorescence-labeled antibody. To confirm cell viability, biotin-labeled anti-MHC class I (H-2K^b) antibodies and TxR-labeled streptavidin were used during primary incubation, inducing cross-linking and internalization of labeled MHC class I molecules. The left part of the figure demonstrates complete staining of acetone-fixed cells as a control. ×1,000. (E) Deglycosylation of m-IC_cl2 cells in situ. Before staining with TLR4 or Con A, cells were treated with Endo H or PNGase F. ×1,000.