TRPC6 is required for LPS-induced NF-κB activation and inflammatory cytokine production. (A) WT mice were exposed to nebulized PBS or LPS for 1 h. Lungs were isolated at the indicated times. Lung lysates were immunoblotted with either phosphoSer536-p65 antibody or ICAM-1 antibody. Densitometric analysis represents mean ± SD of three individual experiments. * indicates significant increase from PBS-exposed group (P < 0.05). (B) WT and Trpc6^−/− lungs were exposed to PBS or 1 mg/ml LPS challenge. Lungs were harvested at the indicated times, and lysates were immunoblotted with either phosphoSer536-p65 antibody or ICAM-1 antibody. Blots were probed with anti-actin antibodies to verify equal protein loading. Densitometric analysis represents mean ± SD of three individual experiments. * indicate values significantly different from values at 0 h (P < 0.05). (C) WT and Trpc6^−/− MLECs were exposed to LPS for indicated times. mRNA was isolated and analyzed for ICAM1 expression by real-time PCR. Data represent mean ± SD of three individual experiments. * indicates values higher than values in WT-MLECs at 0 or 2 h, or values in TRPC6^−/− MLECs after LPS challenge. (D) WT and Trpc6^−/− lungs harvested 4 h after nebulized LPS challenge were homogenized using RIPA buffer. A total of 100 µl of each lung lysate was used for the determination of cytokines using mouse Bio-Plex Multiplex Cytokine Assay kit. Data are expressed as mean ± SD of three independent experiments. * indicates significance from control group (P < 0.05).