FIG. 1.

Activation of ERK1/2 and p38 MAPKs in J774A.1 cells treated with different Brucella strains or with Brucella LPSs. ERK1/2 and p38 MAPKs were determined by Western immunoblotting of J774A.1 cells treated (A) for 30 min with different strains of smooth or rough Brucella (MOI = 40), (B) for different periods of time with B. suis manB or B. suis (MOI = 40), or (C) for 30 min with different concentrations of purified LPSs from smooth B. abortus 2308 (0.050 μg/ml to 5 μg/ml) or rough B. abortus 45/20 (0.025 μg/ml to 2.5 μg/ml). The LPSs were obtained from I. Moriyón (University of Navarra, Pamplona, Spain). They were carefully solubilized by sonication. Their purification and properties have been reported elsewhere (2, 14). The effect of the smooth LPS was assessed at concentrations double those for the rough LPS to compare the lipid A of both LPSs (I. Moriyón, personal communication) (D) or for 30 min with B. suis manB (MOI = 40, 15, and 5) or B. suis (MOI = 40); 2 × 10⁶ J774A.1 cells were cultured at 37°C for the indicated periods of time in 150 μl of RPMI 1640 alone (cells) or supplemented with B. suis, B. melitensis 16 M (B. mel), B. abortus 2308 (B. 2308), B. ovis REO198 (B. ovis), B. suis manB (B. manB), B. melitensis R5 (B. R5), B. melitensis B3B2 (B. B3B2), B. abortus RB51 (B. RB51) or B. abortus 45/20 (B. 45/20), or with different concentrations of the purified LPS from B. abortus 2308 or B. abortus 45/20. They were then rinsed, lysed, and after sodium dodecyl sulfate-10% acrylamide gel electrophoresis and transfer to nitrocellulose membrane, analyzed with phosphospecific antibodies against ERK1/2 or p38 active kinases. The blots were stripped and reprobed with pan antibodies (A and C). In panel B, the same blot was sequentially analyzed for phospho-ERK1/2, phospho-p38, and pan p38 MAPKs. Results are representative of three separate experiments which gave identical results.