Goat anti galectin 9 antibody

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.

Western blot was performed according to standard protocols. In brief, 5 to 10 10 µm thick crysections were suspended in 60 µL Laemlli sample buffer (Biorad) supplemented with 1∶20 β-mercapto-ethanol. Samples were boiled for 5 minutes and immediately separated by gel electrophoresis on a 15% polyacrylamide gel and transferred onto PVDF membranes (Millipore). Membranes were blocked with Oddyssey blocking buffer (LI-COR Biosciences) for 1 hour and incubated overnight at 4°C with either rabbit anti-galectin-1 antibody (Peprotech) or goat anti-galectin-9 antibody (R&D systems). Loading of the gels was checked by α-actin detection using mouse anti-α-actin (1∶10000, MP Biomedicals). The membranes were washed three times with PBS/0.1% tween and subsequently incubated with the appropriate secondary IRDye antibodies (LI-COR Biosciences) at room temperature for 1 hour. Finally, membranes were washed with PBS/0.1% tween and rinsed with PBS after which images were acquired using the Odyssey infrared imaging system (LI-COR Biosciences).

Spot blot analysis was performed with 10 µL drops containing 3 µg of the specific proteins that were spotted onto a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 h, washed with PBS/0.1% Tween, and incubated with 1 µg/mL recombinant galectin-1 or galectin-9 in PBS/0.1% Tween for 1 h. As control, the incubation step with recombinant galectin-1 was omitted. After washing with PBS/0.1% Tween, the membrane was incubated with rabbit anti-galectin-1 antibody (1:1000, Peprotech) or goat anti-galectin-9 antibody (1:250, R&D Systems) for 1 h. Following three washing steps, the appropriate IRDye-labeled secondary antibody (1:10000, LI-COR Biosciences) was applied for 1 h. Finally, the membrane was washed twice with PBS/0.1% Tween and rinsed in PBS. Images were obtained by scanning the membrane with the Odyssey infrared imaging system (LI-COR Biosciences).