Extended Data Fig. 2 |. LT effects on MKK4 and MKK7.

a, C57BL/6J were injected IP with protective antigen (PA) (35 μg) or LT (35 μg) and spleens harvested at 24 h, lysed and analyzed for MeK1, MKK4 and MKK7 cleavage (sequentially, in order shown, re-probing the same blot, using antibodies reactive to N-termini of the proteins). The MeK1 and MKK7 antibodies were detected with an anti-rabbit secondary antibody tagged with same wavelength IR800 label, while MKK4 was detected with an anti-rabbit antibody tagged with an IR680 dye. In the lower panel both secondary antibodies react with all three primary antibodies, resulting in yellow color where both MKK7 and MeK1 are present (lanes 1,2,5), orange where MKK7 alone is present (lanes 3,4, 6) and a lower migrating (red) MKK4 band present equally in all samples (lanes 1–6). b, BMDMs from C57BL/6J mice were treated with LT (1 μg/mL, 1h and 18 h) and analyzed for MKK4 and MKK3 cleavage. The MKK3 band is not seen in spleen lysates, processed as in (a). In panel a MeK1 and MKK7 bands overlap with each other as well as with MKK4, thus densitometry quantification of MKK4 bands was performed only for panel b (see Supplementary Table 1).