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. 2013 Mar 19;8(3):e59301. doi: 10.1371/journal.pone.0059301

Figure 6. HSCARG inhibits p47phox expression through downregulating NF-κB activity.

Figure 6

(A) NF-κB activity was examined by the luciferase report assay in HEK293 cells transfected with 2 µg pRK-Flag-HSCARG or pRK-Flag empty vector for 48 h, in the absence or presence of PMA treatment. (B) The expression of HSCARG was confirmed by western blot analysis using monoclonal anti-Flag antibody. (C) The effects of HSCARG on p65 nuclear translocation and p47phox expression in HEK293 cells treated with or without PMA. HEK293 cells were transfected with pRK-Flag-HSCARG or empty pRK-Flag vector, and then nuclear and cytoplasmic extracts were prepared using Nuclear and Cytoplasmic Extraction Reagents from Thermo Scientific. Extracts were then fractioned by SDS-PAGE and analyzed by immunoblotting using indicated antibodies. Lamin B and α-tubulin were used as nuclear and cytoplasmic loading controls, respectively. (D) The pathway through which HSCARG downregulating ROS production. HSCARG inhibits NF-κB activity, which downregulates the expression of p47phox, the subunit of NADPH oxidase. Attenuated p47phox activity further decreases ROS generation.