Recombinant galectin 9

B cells were treated with 1 μM recombinant galectin-9 (R&D Systems) in 2% BSA in RPMI for 30 min at 37°C. Cells were washed 1X with PBS or RPMI and resuspended in PBS for immediate use in experiments. To stain for rGal9, Daudi cells were first blocked with purified anti-human CD16/CD32 (Fc-block, 1:250 v/v; BD Biosciences, Cat. No 564220) in PBS for 15 min at room temperature. Cells were washed two times and immunostained with goat anti-galectin-9 antibody (R&D Systems, Cat. No. AF3535) at 1:100 v/v for 1 h in 2% BSA in PBS. Cells were washed 3 times, followed by labeling with Cy3-conjugated AffiniPure bovine anti-goat antibody (Jackson Immunoresearch Laboratories, Cat. No. 805-165-180) at 1:1000 v/v in 2% BSA in RPMI for 30 min at 4°C.

A stable form of recombinant galectin-9 was obtained through our collaborators at GalPharma Co., Ltd. (Kagawa, Japan). A natural form of recombinant galectin-9 was purchased from R&D Systems (Minneapolis, MN; catalog # 2045-GA-050). Lck inhibitor A770041 was purchased from Axon Med Chem (Reston, VA). ERK inhibitor LY3214996 was purchased from Selleck Chemicals (Houston, TX). CREB inhibitor 666-15 and InSolution Rapamycin were purchased from Millipore Sigma (Burlington, MA). SMARTpool Accell Lck siRNA was purchased from Dharmacon (Lafayette, CO; cat # E-003151-00-0005) and Silencer™ Select Negative Control No. 1 siRNA was purchased from Thermo Fisher (Waltham, MA; cat# 4390843). ImmunoCult Human CD3/CD28 T Cell Activator was purchased from STEMCELL (Vancouver, BC, Canada).