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Updated: May 4, 2026
Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins
Published on: June 14, 2016
[Main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion]
Peng Wang1, Xiang-dong Zhou2, Juliy M Perelman
1Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Objective:
To explore the main mediated molecules of mucin (MUC) 5AC extracellular secretion stimulated by airway shear stress (SS).
Methods:
The 16 human bronchial epithelial (HBE) cells were cultured and randomized divided by Stata software into 5 groups: A. control group; B. SS stimulated group; C. SS stimulated & NSC23766 (a specific inhibitor of Rac-1) incubated group; D. SS stimulated & Cytochalasin D incubated group; E. Cortactin-siRNA (a small interfering RNA of Cortactin) transfected & SS stimulated group. Each group consisted of 6 parallel wells. Triplicate experiments were performed for statistical analysis. Rhythmic rotating device was used to simulate the breathing air flow mediated shear stress. The function of Cortactin was inhibited by Cortactin-siRNA. The relative content of MUC5AC in supernatant was measured by enzyme linked immunosorbent assay (ELISA). The p-Cortactin (phosphorylation Cortactin) relative level, Cortactin relative level and the effect of transfection were measured with Western blotting. And laser confocal microscope was used to observe the polymerization of F-actin.
Results:
The transfection of Cortactin-siRNA successfully inhibited the function of Cortactin. The relative content of MUC5AC was (0.210 ± 0.013), (0.631 ± 0.025), (0.473 ± 0.112), (0.330 ± 0.067), (0.272 ± 0.019) in groups A, B, C, D and E, the group B was significantly higher than any other group (P = 0.000, 0.043, 0.000, 0.000). The Cortactin relative level in group B (0.670 ± 0.048) was significantly higher than that in group E (0.132 ± 0.014) (P < 0.01). But as compared with groups A, C, D (0.641 ± 0.016, 0.622 ± 0.012, 0.653 ± 0.027), there was no significance (all P > 0.05). The p-Cortactin relative level in group B (0.582 ± 0.067) was significantly higher than that in groups A, C, E (0.131 ± 0.011, 0.393 ± 0.045, 0.170 ± 0.016) (P = 0.000, 0.021, 0.000). But as compared with group D (0.511 ± 0.029), there was no significance (P = 0.246).
Conclusion:
Rac-1, Cortactin and F-actin are the main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion.
