Figure 6.
Figure 6. Intracellular distribution of SI-CLP in human macrophages. Peripheral blood–derived monocytes were stimulated with IL-4 and dexamethasone for 6 days and used for immunofluorescent/confocal microscopy examination. SI-CLP was detected with rat mAb 1C11/antirat Cy3-conjugated secondary ab (red). Other proteins are visualized in green. Merge of green and red is shown in yellow; red and blue, in pink; and green, red, and blue, in white. (A) SI-CLP strongly colocalizes with Lamp1. (B) SI-CLP strongly colocalizes with CD63, marker for secretory lysosomes. (C) SI-CLP is found in p62lck-positive late endosomes. (D) SI-CLP partially colocalizes with stabilin-1 in TGN but is absent from stabilin-1–positive early endosomes. (E-F) Recombinant FLAG-tagged SI-CLP is missorted in globular structures localized in nuclear area (controlled by Dapi) in stably transfected H1299 cells. 1C11-positive structures did not colocalize with lysosomal markers (not shown). Transient overexpression of stabilin-1 resulted in relocalization of SI-CLP into the cytoplasm. Stabilin-1 and SI-CLP partially colocalize with TGN46. (G) MϕIL-4/Dex were transfected with control siRNA and stabilin-1 siRNA on day 4 of culture. Intracellular distribution of SI-CLP was analyzed on day 6 (2 days after transfection). The decreased sorting into lysosomes and abnormal concentration in nuclear rim structures were detected in part of cells transfected with stabilin-1 siRNA. (H) Stabilin-1 protein expression in MϕIL-4/Dex is efficiently suppressed by stabilin-1 siRNA. Western blot analysis with anti–stabilin-1 F4 ab. (I) Western blot analysis of SI-CLP secretion in long-term macrophage cultures. SI-CLP is detected in conditioned medium of IL-4–stimulated macrophages after 18 and 21 days of stimulation. Costimulation with dexamethasone results in intracellular accumulation of SI-CLP but blocks its secretion. The absence of cell damage was controlled by quantification of lactate dehydrogenase (LDH) activity in conditioned medium using a cytotoxicity detection kit (Roche, Mannheim, Germany).

Intracellular distribution of SI-CLP in human macrophages. Peripheral blood–derived monocytes were stimulated with IL-4 and dexamethasone for 6 days and used for immunofluorescent/confocal microscopy examination. SI-CLP was detected with rat mAb 1C11/antirat Cy3-conjugated secondary ab (red). Other proteins are visualized in green. Merge of green and red is shown in yellow; red and blue, in pink; and green, red, and blue, in white. (A) SI-CLP strongly colocalizes with Lamp1. (B) SI-CLP strongly colocalizes with CD63, marker for secretory lysosomes. (C) SI-CLP is found in p62lck-positive late endosomes. (D) SI-CLP partially colocalizes with stabilin-1 in TGN but is absent from stabilin-1–positive early endosomes. (E-F) Recombinant FLAG-tagged SI-CLP is missorted in globular structures localized in nuclear area (controlled by Dapi) in stably transfected H1299 cells. 1C11-positive structures did not colocalize with lysosomal markers (not shown). Transient overexpression of stabilin-1 resulted in relocalization of SI-CLP into the cytoplasm. Stabilin-1 and SI-CLP partially colocalize with TGN46. (G) MϕIL-4/Dex were transfected with control siRNA and stabilin-1 siRNA on day 4 of culture. Intracellular distribution of SI-CLP was analyzed on day 6 (2 days after transfection). The decreased sorting into lysosomes and abnormal concentration in nuclear rim structures were detected in part of cells transfected with stabilin-1 siRNA. (H) Stabilin-1 protein expression in MϕIL-4/Dex is efficiently suppressed by stabilin-1 siRNA. Western blot analysis with anti–stabilin-1 F4 ab. (I) Western blot analysis of SI-CLP secretion in long-term macrophage cultures. SI-CLP is detected in conditioned medium of IL-4–stimulated macrophages after 18 and 21 days of stimulation. Costimulation with dexamethasone results in intracellular accumulation of SI-CLP but blocks its secretion. The absence of cell damage was controlled by quantification of lactate dehydrogenase (LDH) activity in conditioned medium using a cytotoxicity detection kit (Roche, Mannheim, Germany).

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