Fig. 3.

Upregulation of SOCS1 after TLR7 and TLR9 costimulation. (A) RAW264.7 cells were stimulated with either an agonist for TLR7 or TLR9 or both for the indicated time. The protein expression in the total lysates was analyzed by Western blotting using antibodies against A20, IRAK-M, SOCS-1 and β-actin. (B) The mRNA expression levels of A20, SOCS-3, SOCS-1, IRAK-M and β-actin were determined through RT-PCR. (C) RAW264.7 cells were transfected with siRNA targeting SOCS-1 mRNA or scrambled siRNA and stimulated with either an agonist for TLR7 or TLR9 or both overnight (1st). After stimulation, the cells were washed with PBS and incubated with culture media overnight. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd) and the production of TNF-α was measured through ELISA.