Role of MYLK as a TRPC6 effector mediating increased lung vascular permeability and NF-κB activation. (A) WT and Mylk^−/− lungs were perfused with vehicle or 100 µM OAG for 20 min, after which K_f,c was determined. Bar graph shows mean ± SEM of data from three experiments. * indicates significant increase in K_f,c values compared with vehicle perfused WT lungs or Mylk^−/− lungs perfused with vehicle or OAG (P < 0.05). (B) Lungs from WT and Mylk^−/− mice were harvested 4 h after PBS or 1 mg/ml LPS challenge. Lung lysates were immunoblotted with either phosphoSer536-p65 antibody or ICAM-1 antibody. Blots were probed with anti-actin antibodies to verify equal protein loading. Data represent results from three separate experiments. * indicates values different from values under basal conditions (no LPS; P < 0.05). (C and D) LPS exposed WT and Mylk^−/− mice were injected with Evans blue–labeled albumin retroorbitally 30 min before termination of experiments and lung wet/dry ratio (C) and albumin extravasation from lungs (D) were determined. Data represent mean ± SEM of three individual experiments. * indicates significant increase from PBS-exposed group (P < 0.05). (E) Lungs from WT, Trpc6^−/−, and Mylk^−/− mice were harvested 4 h after PBS or 1 mg/ml LPS challenge. Lung lysates were immunoblotted with anti-phospho MLC antibody. Blots were stripped and reprobed with anti-MLC antibodies to verify equal protein loading. Data represent results from three separate experiments. * indicates values that are significantly higher between control versus LPS treatment (P < 0.05). (F, left) HPAECs were transduced either with dominant-negative TRPC6 (DN-TRPC6) mutant or empty vector for 24 h. Cells were loaded with Fura 2-AM. After 15 min, cells were stimulated with 100 µM OAG in 2 mM Ca²⁺ buffer and changes in intracellular Ca²⁺ in response to OAG were determined ratiometrically. Each representative trace is the average response of 20–25 cells, and these experiments were repeated three times. (right) HPAECs transducing DN-TRPC6 mutant or kinase-dead MYLK (KD-MYLK) mutant for 24 h were stimulated with 1 µg/ml LPS for 4 h. Cell lysates were immunoblotted with phosphoSer536-p65, ICAM-1 or phosphorMLC antibodies. Blots were probed with anti-actin antibodies to verify equal protein loading. Densitometric analysis was performed on data from three separate experiments. * indicates values different from values under basal conditions (no LPS; P < 0.05).