Extended Data Fig. 2: TGFβ pathway-related genes are not altered in Havcr2^cKO microglia.

a, The structure of Dox-inducible (Tet-on system) Flag-conjugated HAVCR2 plasmid, which was used for CoIP-MS screening for binding partners of Tim-3. b, Tim-3 expression was evaluated by western blot in Jurkat cells transduced with Dox-inducible Flag-conjugated HAVCR2, after treatment with Dox. Supernatant samples were from the supernatant solution after incubation of input cell lysate with anti-Flag antibody-conjugated dynabeads. c, d, Proximity ligation assay for Tim-3, Smad2, and Tgfbr2 in primary microglia from the brain (c) and the spinal cord (d) of WT mice. e, pSmad2, Smad2, and Gapdh were quantified in Havcr2^KO primary microglia by western blotting. The samples for the control Gapdh were run on a separate gel as sample processing controls (Supplementary Fig. 1 for gel source data). f, pSmad3 and Smad3 expression was quantified in Havcr2^cKO microglia by flow cytometry (n = 5 control, 4 Havcr2^cKO, independent mice). g, Microglia (live CD45^intCD11b⁺) were sorted from control and Havcr2^cKO male mice and cultured in the presence of M-CSF (10 ng/mL), GM-CSF (10 ng/mL), or TGFβ (10 ng/mL) for 16 h. TGFβ pathway-related gene expressions were quantified by RT-qPCR (n = 3/condition, 2 for Havcr2^cKO-GM-CSF group, independent samples. h, HEK293 cells were transfected with WT-Havcr2 plasmid. Smad3 and pSmad3 expression was quantified by flow cytometry (n = 4/condition, independent samples). i, Tgfbr2^KO BV2 cells were stimulated with TGFβ, and evaluated for pSmad2 expression by flow cytometry. j, Smad2 was quantified in HEK293 cells by flow cytometry after transfection with WT and mutant Havcr2 constructs (n = 3/condition, independent samples). k, HEK293 cells were transfected with WT-Havcr2 plasmid and either non-phosphorylated Smad2 mimetic (3SA) or phosphorylated Smad2 mimetic (2 SD) plasmid. The cell lysate was coimmunoprecipitated for HA-Tim-3 and blotted for Smad2 mimetics. The samples for the control β actin were run on the same gel as loading controls (Supplementary Fig. 1 for gel source data) l, Microglia (live CD45^intCD11b⁺) were sorted and cultured in the presence of Galectin 9 (3 ug/mL), or CEACAM1 (3 ug/mL), combined with TGFβ (1 ng/mL) for 16 h. pSmad2 expression was quantified by flow cytometry (n = 3/condition, independent samples). m, HEK293 cells were transfected with WT-Havcr2 plasmid and either one of three plasmids with major Smad2 domains: MH1 (Amino acid sequences (AA) 1-181), and MH2 (AA 274-467). The cell lysate was co-immunoprecipitated for HA-Tim-3 and blotted for Smad2 domains. The samples for the control β actin were run on the same gel as sample processing controls (Supplementary Fig. 1 for gel source data). Dox, doxycycline; Sup, supernatant; CoIP, coimmunoprecipitation; MS, mass spectrometry. Results are shown from one experiment, representing at least two independent experiments for h-m. Data are mean ± s.e.m.

Source data