Fig 1: TIMP-1–CD74 interaction induces ZAP-70 signaling.A, C, D, and E, representative Western blots of cell lysates from B lymphoma cells treated as indicated. Levels of phosphorylated ZAP-70 and GAPDH were analyzed. A, cells were treated with 500 ng/ml human recombinant WT TIMP-1 (n = 3), 100 ng/ml human recombinant MIF (n = 6) or were left untreated (n = 4) (left). Fold change of ZAP-70 activation was determined by densitometric analysis of band intensities normalized to levels of GAPDH (right). B, representative flow cytometric analysis of CD74 expression by three different CD74 knockdown B lymphoma cells (shCD74 #1, #2, #3) or control cells (shNT). Contour plots show the frequency of CD74-positive cells. C, representative Western blots of cell lysates from three different CD74 knockdown B lymphoma cells (shCD74 #1, #2, #3) or control cells (shNT) treated as indicated. D, B lymphoma cells were stimulated with 500 ng/ml human recombinant TIMP-1 (n = 5), equimolar human recombinant N-TIMP-1 (n = 4), or left untreated (n = 5) (left). Fold change of ZAP-70 activation was determined by densitometric analysis of band intensities normalized to levels of GAPDH (right). E, B lymphoma cells were stimulated with 500 ng/ml TIMP-1 after preincubation with or without an anti-CD74 blocking peptide. A, C, and D, results are represented as the mean ± SD. For statistical analyses, a one-sample t test was used in case of normal distribution or a one-sample Wilcoxon test in the absence of normal distribution, ∗p ≤ 0.05; n.s., not significant. MIF, macrophage migration inhibitory factor; N-TIMP-1, N-terminal domainof TIMP-1; TIMP-1, tissue inhibitor of metalloproteinases-1; ZAP-70, zeta chain–associated protein kinase-70.
Fig 2: In silico prediction of TIMP-1–CD74 interacting residues by molecular docking.A, illustration of the molecular docking approach to predict the interaction site of TIMP-1 (PDB ID: 1uea) with CD74 (PDB ID: 1ile). Known crystal structures are shown in surface depiction, and dashed lines indicate unresolved structural regions. B, representative complexes of the three energetically most favorable TIMP-1–CD74 clusters obtained from the haddock docking approach and illustrated with UCSF Chimera. The N-terminal domain of TIMP-1 is shown in light blue, the C-terminal domain is shown in blue, and the CTC motif of TIMP-1 is shown in orange. Monomer 1 of the CD74 homotrimer is shown in dark green, monomer 2 is shown in light green, and monomer 3 is shown in yellow. The indicated score represents the respective calculated Haddock score, an indicator of the intermolecular energy of the respective complexes. C, surface representation of TIMP-1 with interface regions participating in the interaction with CD74 highlighted in red. The CTC motif is highlighted in orange (top). TIMP-1 molecules were turned around the x, y, and z axes as indicated. D, surface representation of CD74 with interface regions participating in the interaction with TIMP-1 highlighted in red. CD74 molecules were turned around the x, y, and z axes as depicted.
Fig 3: Expression of MIF-related receptors in H1 and H9 cells.A. The expression of MIF-related receptors in human H1 and H9 embryonic stem cells was detected by ICF; DAPI stained nuclei showed blue fluorescence, where the depth of red fluorescence indicates the MIF-related receptors CD74, CD4, CD44, CXCR2, CXCR4 and CXCR7, the deeper the red, the denser the distribution, indicating the higher the related receptor expression level, with a magnification of 1200 and a scale of 10m; B. Western blot was used to detect the expression of MIF-related receptors in human H1 and H9 embryonic stem cells; C. Semi-quantitative analysis of protein expression, compared with Tubulin, ****P <0.0001, n = 3.
Fig 4: TIMP-1 interacts with CD74.A, qRT-PCR analysis of 18S rRNA and CD74 RNA expression in control (SFempty) and CD74-overexpressing (SFCD74) LX-2 cells. Results are represented as the mean ± SD of three technical replicates. B, representative Western blot detecting human CD74 in control (SFempty) and CD74-overexpressing (SFCD74) LX-2 cells. Equal protein loading was examined by whole-protein staining by SYPRO Ruby protein stain. C, representative contour plots showing cell populations of SFempty and SFCD74 LX-2 cells after exclusion of cell debris and dead cells. Fluorescence-minus-one (FMO) controls and CD74-FITC stained cells are shown, respectively. CD74-FITC fluorescence intensity (y-axis) is plotted against forward scatter (FSC) values. Indicated CD74-positive gates were set in FMO controls of each cell line. D, representative coimmunoprecipitation of CD74 and TIMP-1. Agarose beads conjugated with anti-CD74 antibodies or control IgG-conjugated beads were incubated with the nondenatured cell lysate derived from CD74 overexpressing cells, and protein abundance in pull-down fractions was analyzed via Western blot. E, representative confocal image of CD74-overexpressing LX-2 cells stimulated with fluorescently labeled recombinant human TIMP-1 (Alexa555-TIMP-1). The nucleus is shown in blue, Alexa555-TIMP-1 is shown in magenta, and CD74 is shown in green. Regions of TIMP-1–CD74 colocalization are shown in gray. The scale bars represent 3 μm. TIMP-1, tissue inhibitor of metalloproteinases-1.
Fig 5: In vitro validation of the in silico–predicted docking site of TIMP-1 with CD74.A, representative confocal image of CD74-overexpressing LX-2 cells stimulated with fluorescently labeled recombinant human N-TIMP-1 (Alexa555N-TIMP-1). The nucleus is shown in blue, Alexa555N-TIMP-1 is shown in magenta, CD74 is shown in green. Regions of N-TIMP-1–CD74 colocalization are shown in gray. The scale bars represent 4 μm. B, representative dot blot assay showing the binding of endogenous CD74 derived from nondenatured B lymphoma cell lysates (shNT or shCD74) to immobilized TIMP-1 or MIF, respectively. Binding of cell lysate–derived CD74 to uncoated (shNT: n = 10, shCD74: n = 5), TIMP-1-coated (shNT: n = 10, shCD74: n = 5), and MIF-coated (shNT: n = 9, shCD74: n = 5) dots was determined by immunoblotting against CD74 (left) and quantified using densitometric analysis of dots (right). C, representative confocal image of a B lymphoma cell stimulated with fluorescently labeled recombinant human TIMP-1 (Alexa555TIMP-1). Distribution of TIMP-1 (magenta), CD74 (green), as well as TIMP-1–CD74 colocalization (white) is shown. Surfaces of TIMP-1–CD74 colocalization are displayed in gray, and the nucleus is shown in blue. The scale bars represent 2 μm. D, representative dot blot assay investigating N-TIMP-1 binding to CD74. Immobilized N-TIMP-1 was incubated with nondenatured cell lysate from B lymphoma cells. Binding of cell lysate-derived CD74 to uncoated (n = 5) and N-TIMP-1-coated dots (n = 5) was determined by immunoblotting against CD74 (left panel) and quantified using densitometric analysis of dots (right panel). B and D, results are represented as the mean ± SD. For statistical analyses, a one-sample t test was used in case of normal distribution or a one-sample Wilcoxon test in absence of normal distribution, ∗p ≤ 0.05. MIF, macrophage migration inhibitory factor; N-TIMP-1, N-terminal domain of TIMP-1; TIMP-1, tissue inhibitor of metalloproteinases-1.
Supplier Page from Abcam for Anti-CD74 antibody [EPR4064]