Figure 1.
Characterisation of 2D9, a monoclonal anti-NOD2 antibody. (A, B) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express HA tagged Nod2 (114 kD), HA tagged Nod1 (110 kD), HA tagged Ipaf (118 kD), Myc tagged Apaf-1 (138 kD), Flag tagged RICK (63 kD), or Flag tagged Pypaf-1 (120 kD). Cell lysates were immunoblotted with 2D9, a mouse monoclonal anti-NOD2 antibody (A), mouse monoclonal anti-HA antibody (16B12), mouse monoclonal anti-Flag antibody (M2), or mouse monoclonal anti-Myc antibody (9E10) (B). (C) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express HA tagged wild-type Nod2 or mutant Nod2 lacking caspase recruitment domains (ΔCARDs) or expressing CARDs alone (CARDs). Immunoblottings with 2D9 or anti-HA antibody are shown. (D) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express wild-type Nod2 or mutant Nod2, P268S, P268S/R702W, P268S/G908R, or P268S/L1007fs. Cell lysates were blotted with 2D9. (E) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express wild-type Nod2. Cell lysates were immunoprecipitated with 2D9 or M2, an isotype matched control antibody. Immunorecipitates were blotted with rabbit polyclonal anti-Nod2 antibody. (F) HL60 cells were cultured with or without tumour necrosis factor α (TNF-α) for 24 hours. Cell lysates from 2×108 cells were immunoprecipitated with 2D9 or M2. Immunoprecipitates were blotted with rabbit polyclonal anti-Nod2 antibody.