Journal: European Journal of Immunology
Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans
Figure Lengend Snippet: TIM‐3 and CEACAM1 do not interact in trans . (A) Binding of indicated Ig fusion proteins to immobilized recombinant galectin‐9 (Gal‐9) and CEACAM1 proteins was analyzed by ELISA. Results are representative for four independently performed experiments. Information on the used proteins is provided in the material and method section. (B) Jurkat cells transduced to express ICOS, TIM‐3, or CEACAM1 were probed with the respective antibodies and analyzed by flow cytometry (gray histograms). Open histograms show the reactivity of control Jurkat cells. Bottom: results of a representative binding experiment of indicated Ig fusion proteins used at a final concentration of 10 μg/mL to control Jurkat cells or Jurkat cells expressing ICOS, TIM‐3, or CEACAM1. Binding was detected via flow cytometry by an APC‐conjugated goat‐anti‐human IgG (Fc‐specific) Ab. (C) Binding of indicated Ig fusion proteins (final concentrations: 31.6, 10, 3.16, 1, and 0.316 μg/mL) to control Jurkat cells and Jurkat cells expressing ICOS, TIM‐3, or CEACAM1. Binding was detected as described in (B). Results are shown from three different experiments performed in duplicates. Binding signals (gMFI) were normalized to background binding (gMFI values obtained with secondary reagent only). (D) Binding of CEACAM1‐Ig (final concentration: 10 μg/mL) to control Jurkat cells and CEACAM1‐expressing Jurkat cells in absence or presence of a blocking CEACAM1 mAb. Results are shown from four different experiments performed in duplicates. (E) Cells coexpressing eCFP or eGFP and the indicated cell surface molecules were preincubated for 1 h and conjugate formation between cells expressing different fluorescent proteins was assessed via flow cytometry. (F) Results of three independently performed cell–cell binding assays are summarized. (A, C, D, and F) Standard deviation is shown.
Article Snippet: TIM‐3 and CEACAM1 Ig fusion proteins (TIM‐3‐Ig, CEACAM1‐Ig) were purchased from SinoBiological (SB; People's Republic of China).
Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay, Expressing, Blocking Assay, Standard Deviation