Mouse Anti-LGALS9 Recombinant Antibody (clone TIB-141)

A, RBL-2H3 cells cultured in 96-well plates (2× 10⁴ cells/well) were stimulated by the addition of TIB-141 and TNP-BSA. hG9Null and mG9Null were added 10 min before the stimulation. β-HEX activity was determined in the culture medium, and a cell lysate was prepared 60 min after the stimulation. The horizontal broken line represents the mean value for a control sample (without TIB-141 and TNP-BSA). Lactose was used at a concentration of 20 mM. The data shown in A are derived from a single representative experiment of two independent experiments. The results are presented as means±S.D.(error bars) (n=3). B, RBL-2H3 cells cultured in 12-well plates (2.5×10⁵ cells/well) were treated. hG9Null and mG9Null were used at 0.3 and 1 μM, respectively. A whole cell lysate was prepared at the time points indicated after the addition of TIB-141 and TNP-BSA. Samples were electrophoretically separated in SDS-12.6% polyacrylamide gels under reducing conditions (5 μg/lane), blotted onto PVDF membranes, and then subjected to immunodetection using anti-phospho-p44/42 MAPK (Erk1/2) (Thr²⁰²/Tyr²⁰⁴), anti-p44/42 MAPK, and anti-actin antibodies.

Nakakita, S. I., Itoh, A., Nakakita, Y., Nonaka, Y., Ogawa, T., Nakamura, T., & Nishi, N. (2016). Cooperative interactions of oligosaccharide and peptide moieties of a glycopeptide derived from IgE with galectin-9. Journal of Biological Chemistry, 291(2), 968-979.

HPLC peak fractions derived from 3.0 nmol (A and B) or 15 nmol (C) of PE-TIB-141 were examined for their ability to antagonize G9Null. The peak fractions were lyophilized and then dissolved in 50 μl of PBS. The peptide solutions (25 μl) were mixed with G9Null solutions (225 μl), followed by 30-min incubation at room temperature before adding to the RBL-2H3 cell culture. The data are derived from a single representative experiment of two independent experiments. The results are presented as means±S.D. (error bars) (n=3).

Nakakita, S. I., Itoh, A., Nakakita, Y., Nonaka, Y., Ogawa, T., Nakamura, T., & Nishi, N. (2016). Cooperative interactions of oligosaccharide and peptide moieties of a glycopeptide derived from IgE with galectin-9. Journal of Biological Chemistry, 291(2), 968-979.

A, P4 –P23 preparations were incubated overnight at 37 °C in the absence (C) or presence (G) of peptide-N-glycosidase F and then analyzed by SDS-PAGE (13.5% polyacrylamide gel) under reducing conditions. The gels were stained with Coomassie Brilliant Blue R-250. B, the selected band materials subjected to N-terminal amino acid sequence analysis are indicated. C, MALDI-TOF mass spectrum of the P23 preparation treated with peptideN-glycosidase F. The sample was applied onto a ground steel target using sinapinic acid as a matrix.

Nakakita, S. I., Itoh, A., Nakakita, Y., Nonaka, Y., Ogawa, T., Nakamura, T., & Nishi, N. (2016). Cooperative interactions of oligosaccharide and peptide moieties of a glycopeptide derived from IgE with galectin-9. Journal of Biological Chemistry, 291(2), 968-979.