B7-1 is not induced in immortalized podocytes after injury. (a) B7-1 mRNA is barely detectable in immortalized podocytes at baseline and is not induced after lipopolysaccharide (LPS) stimulation. IL-6 mRNA confirmed Toll-like receptor 4 engagement after LPS treatment. (b) Western blot of B7-1 (SC-9091 from Santa Cruz Biotechnology Inc.) in immortalized podocytes exposed to either vehicle or 50.0 μg/ml LPS for 24 hours. C+: positive control, HeLa cells transfected with a vector containing mouse B7-1 DNA. (c) Coimmunostaining for nestin (red), nephrin (green), and 3 different anti-B7-1 antibodies (purple, 2A2: ab86473 from Abcam [Paris, France]; AF140: from R&D Systems [Lille, France]; and H-208: SC-9091 from Santa Cruz Biotechnology Inc.) in immortalized podocytes 24 hours after exposure to either vehicle (upper panel) or 50.0 μg/ml LPS (lower panel). Bar = 10 μm. The data are represented as the means ± SEM. Analysis of variance followed by a Tukey-Kramer test; podocytes treated with vehicle versus LPS: *** P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.