Pd 1 d4w2j xp rabbit mab

Jurkat T cells (1 × 10⁶) overexpressing PD-1 or/and TIM-3 were treated with or without 2 μg/ml recombinant Gal-9 in cell culture medium for 2 h. Cells were then collected and lysed in 0.1 ml RIPA buffer for 15 mins at 4 °C. The cell lysates were centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant was collected in new microtubes, and the pellet was lysed in SDS sample buffer. Proteins in the supernatant and pellet fractions were then analyzed by western blotting. Mouse anti-Gal-9 antibody clone OTI1G3 (Bio-Rad), PD-1 (D4W2J) XP rabbit mAb (Cell Signaling Technology), and rabbit anti-TIM-3 antibody MAB23652 (R&D Systems) were used at 1:1000, 1:1000, and 2 µg/ml, respectively.

Jurkat cells that express 3×FLAG-tagged bait proteins were lysed in PTY buffer (50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 10 mM Na₄P₂O₇. pH to 7.4) and lysates were incubated for 2 h with anti-FLAG (M2) magnetic beads (Sigma). Beads were then wash 3 times with 5× TBS-T buffer and once with 1× TBS-T buffer. Bound proteins were eluted in SDS-sample buffer and subjected to western blot with specific antibodies. Mouse anti-Gal-9 antibody clone OTI1G3 (Bio-Rad), PD-1 (D4W2J) XP rabbit mAb (Cell Signaling Technology), and rabbit anti-TIM-3 antibody MAB23652 (R&D Systems) were used at 1:1000, 1:1000, and 2 µg/ml, respectively. Where indicated, cells were incubated with 2 μg/ml Gal-9 in PBS at 4 °C for 1 h before lysis.