a– c, Interaction between TIM-3–Ig fusion protein and membrane protein of 60 kDa after deglycosylation derived from surface-biotinylated TK-1 cells. TIM-3–Ig fusion proteins and human IgG-precipitated proteins were deglycosylated by PNGase F and separated by SDS–PAGE. TIM-3–Ig-binding membrane proteins detected by immunoblot. A 60-kDa membrane protein (red circles) and 32-kDa protein consistent with galectin-9 (black circles) are found specifically associated with soluble (s) TIM-3–Ig fusion protein ( a, lane 5) and full-length (f) TIM-3–Ig proteins ( b, lane 5), but not with the pre-clear controls (lanes 3 and 4) or human IgG (lanes 2 and 6). c, sTIM-3–Ig and full-length (fl)TIM-3–Ig interacting proteins were de-glycosylated by PNGase F and separated by SDS–PAGE. Proteins detected by silver staining. A band of 60 kDa (red circle) is specifically associated with sTIM-3–Ig proteins (lane 2), but not with human IgG (lane 5), or TIM-1–Ig or TIM-4–Ig (lanes 3, 4, 6–10). d, Superimposition of previously described IgV-like domains of mCEACAM1 and mTIM-3 demonstrate structural similarity with a score of 2.42 by the structural alignment and root mean square deviation (r.m.s.d.) calculated by Pymol. e, Sequence alignment of the IgV-like domains of mCEACAM1 and mTIM-3 on the basis of the secondary structure alignment in d. f, Sequence alignments of IgV domain sequences of CEACAM1 and overall mTIM and hTIM family members. α helices (orange) and β strands (blue) denoted as underlined segments in hCEACAM1 and mTIM-3. β strands labelled with upper- and lower-case letters for hCEACAM1 and mTIM-3, respectively. Conserved residues are shaded red. Mutated residues are shaded violet for hCEACAM1, and green for hTIM-3. Asterisk (*) indicates positions having a single, fully conserved residue; a dagger ( †) indicates conservation between groups of weakly similar residues; a double-dagger ( ‡) indicates conservation between groups of strongly similar residues. g, Computational modelling as defined by energy calculations (score) relative to r.m.s. values of docking models to define potential cis and trans interfaces between mCEACAM1 and mTIM-3 as described in Supplementary Information and amino acids involved. h, CEACAM1 expression on mouse fibroblast 3T3 cells used to identify a galectin-9-independent ligand. i, CEACAM1 expression on mouse TK-1 cells as in a– c. Representative of three ( a– c, h, i) independent experiments.