Journal: The Journal of Biological Chemistry

Article Title: Galectin-1 Regulates Tissue Exit of Specific Dendritic Cell Populations *

doi: 10.1074/jbc.M115.644799

Figure Lengend Snippet: Galectin-1 binds CD43 on iDCs and tDCs. A, iDCs and tDCs were incubated with recombinant galectin-1 for 1 h at 4 °C. iDCs and tDCs were biotinylated, incubated with recombinant galectin-1 for 1 h at 4 °C, and fixed with DTSSP. After chemical cross-linking, galectin-1 plus bound cell surface counter-receptors were immunoprecipitated ( IP ) with rabbit anti-galectin-1 pAb. Control samples were immunoprecipitated with rabbit serum ( rb serum ). Immunoprecipitates were separated on a 4–12% BisTris polyacrylamide gel in MOPS buffer. Blots were probed with streptavidin-horseradish peroxidase ( HRP ), stripped, and re-probed with antibody against CD43. A single band of ∼115 kDa was detected in samples from both iDCs and tDCs that reacted with the pan-specific CD43 mAb DF-T1. B, iDCs bind slightly more exogenous galectin-1 than tDCs. Recombinant biotinylated galectin-1 was added to iDCs and tDCs at 4 °C, and bound galectin-1 was detected with FITC-conjugated streptavidin. Data shown are representative of four independent experiments. Filled histogram is the rabbit serum ( rb serum ) control staining. C, differences in CD43 core 2 O- glycosylation on iDCs and tDCs were identified by flow cytometry using mAb 1D4 ( left ) that recognizes core 2 O- glycans on human CD43. Although iDCs and tDCs expressed equivalent levels of total CD43, detected by mAb DF-T1 ( right ), iDCs express significantly more CD43 decorated with core 2 O- glycans compared with tDCs. Filled histograms are isotype controls. D, β(1,6)- N -acetylglucosaminyltransferase ( C2GnT-I ) mRNA expression was analyzed by quantitative RT-PCR and normalized to expression of the housekeeping gene 36B4 . Results are from three independent experiments ± S.E. ***, p = 0.001. iDCs express >3-fold more β(1,6)- N -acetylglucosaminyltransferase mRNA than tDCs, consistent with the higher expression of core 2 O- glycan modified CD43 on iDCs compared with tDCs in C .

Article Snippet: The following antibodies were used: rabbit anti-human galectin-1 polyclonal antibody serum (pAb) (Strategic); rat anti-mouse galectin-3 antibody (clone M3/38) (BioLegend); mouse anti-human galectin-9 (Novus Biologicals); mouse anti-human podoplanin (clone D2-40) (Covance); mouse anti-human CD86-phycoerythrin (PE) (clone BU63) (Invitrogen); mouse anti-human CD40-PE (clone HB14) (BioLegend); mouse anti-human CD43 (clone 1D4) (MBL); mouse anti-human CD43 (clone DF-T1) (DakoCytomation).

Techniques: Incubation, Recombinant, Immunoprecipitation, Staining, Flow Cytometry, Expressing, Quantitative RT-PCR, Modification

Journal: The Journal of Biological Chemistry

Article Title: Galectin-1 Regulates Tissue Exit of Specific Dendritic Cell Populations *

doi: 10.1074/jbc.M115.644799

Figure Lengend Snippet: Inhibition of O- glycosylation reverses galectin-1 inhibition of iDC migration and prevents CD43 clustering on iDCs in the presence of galectin-1. A, iDCs were treated with 2 m m Bn-α-GalNAc ( iDC + Bn -α- GalNAc ) or with buffer control ( iDC ). Expression of CD43 bearing core 2 O- glycans was analyzed by flow cytometry with the mAb 1D4. Filled histogram is the isotype control. Treatment with Bn-α-GalNAc decreased binding of mAb 1D4. B, in vitro migration assays were performed as described in C . Bn-α-GalNAc-treated and untreated iDCs were migrated across Matrigel TM plus LECs in the presence or absence of recombinant galectin-1 in Matrigel TM . Inhibition of O- glycan elongation reversed the inhibitory effect of galectin-1 on iDC migration. Results are shown as % migration in the presence of galectin-1 in Matrigel TM /% migration in the absence of galectin-1 in Matrigel TM . Results are representative of three independent experiments. Three replicate samples ± S.D. are shown for each data point, *, p = 0.019. C , iDCs or tDCs were treated with recombinant galectin-1 for 1 h at 37 °C. After fixation, samples were processed for confocal microscopy using antibody against CD43 (mAb DF-T1) and FITC-coupled secondary antibody. Galectin-1 clustered CD43 on iDCs ( white arrows ) but not on tDCs. Nuclei were visualized with DAPI. Two different images from different experiments are shown for each condition. Scale bar, 5 μm. D, iDCs or tDCs were treated with 2 m m Bn-α-GalNAc before recombinant galectin-1 was added for 1 h at 37 °C. Samples were processed as above. Inhibition of O- glycan elongation with Bn-α-GalNAc abrogated galectin-1-induced clustering of CD43 on iDCs. Two different images from different experiments are shown for each condition. Scale bar, 5 μm.

Article Snippet: The following antibodies were used: rabbit anti-human galectin-1 polyclonal antibody serum (pAb) (Strategic); rat anti-mouse galectin-3 antibody (clone M3/38) (BioLegend); mouse anti-human galectin-9 (Novus Biologicals); mouse anti-human podoplanin (clone D2-40) (Covance); mouse anti-human CD86-phycoerythrin (PE) (clone BU63) (Invitrogen); mouse anti-human CD40-PE (clone HB14) (BioLegend); mouse anti-human CD43 (clone 1D4) (MBL); mouse anti-human CD43 (clone DF-T1) (DakoCytomation).

Techniques: Inhibition, Migration, Expressing, Flow Cytometry, Binding Assay, In Vitro, Recombinant, Confocal Microscopy