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. 2019 Sep 18;10:2147. doi: 10.3389/fimmu.2019.02147

Figure 6.

Figure 6

Itch-independent Txnip degradation promotes TLR2-mediated NO production. (A) RAW264.7 cells stably expressed control luciferase shRNA (shLuc) and Itch shRNA (shItch). The expression of Itch and β-actin was measured. Western blotting showed Itch and Txnip expression in WT, shLuc-, and shItch-expressed RAW264.7 cells (B) infected with the indicated MOIs of GAS at 2 h postinfection or (C) stimulated with HK-GAS (MOI 10), LTA (5 μg/ml), and PGN (5 μg/ml) for 2 h. GAPDH was used as an internal control. (D) Txnip expression was detected in cells transiently transfected with a dominant-negative mutant of Itch (C832A) followed by HK-GAS, LTA, and PGN treatment for 2 h. GAPDH is shown as an internal control. The expression ratios of Txnip and Itch to internal controls are shown. (E) shLuc- or shItch-expressing (upper panel) and control plasmid- or ItchC832A-expressing (lower panel) RAW264.7 cells were treated with HK-GAS, LTA, and PGN for 24 h. NO production was measured and shown as the means ± SD of triplicate cultures. ***p < 0.001 compared with shLuc-expressed cells. Protein molecular weights (MW) are indicated in kilodaltons. Western blot results represent at least two independent experiments.