Figure 2

rLsa21 induced cytokine secretion in mouse macrophages.

(A) RAW264.7 cells were incubated with LPS (500 ng/ml) or PAM3CSK (20 ng/ml) or rLsa21 (0.1, 0.5, 1 or 2 μg/ml) with polymixin B for 24 hrs and supernatant was collected to measure levels of TNF-α and IL-6 by ELISA. rLsa21 (2 μg/ml) pre-treated with PK followed by heating at 95 °C for 15 min, or pre blocking of RAW264.7 cells with TLR2/TLR4 monoclonal Antibodies (Biolegend) was used as controls. (B) TLR2^−/−, TLR4^−/− or TLR2^−/−/TLR4^−/− DKO macrophages cell lines were treated with rLsa21 (2 μg/ml), LPS (500 ng/ml) or PAM3CSK (20 ng/ml) and levels of IL-6 and TNF-α in the supernatants were measured with ELISA. Data are representative of three different experiments. Significant differences were calculated using the Student’s t-test (*Indicates P < 0.05). UI indicates uninduced or unstimulated cells, GST- indicates Glutathione S-transferase protein (2 μg/ml) PAM indicates PAM3CSK (20 ng/ml) LPS is E. coli Lipopolysaccharide (500 ng/ml), PK is Proteinase K, PB is Polymixin B (10 μg/ml) and TLR is Toll like receptors.