Immediately after isolation, lungs were lysed in RIPA with 2X HALT protease and phosphatase inhibitors. As a positive control for TRPV4 expression, adult mouse lung epithelial cells (MLE12) (ATCC, Manassas, VA) were routinely cultured in HITES medium (as per formulation suggested by ATCC) with 1% PS, and similarly lysed. Protein was homogenized, quantified using a detergent compatible Lowry assay, and denatured at 95°C in LDS sample buffer containing DTT (CBS Scientific). Protein was loaded at 20 μg per lane into a 4–12% TEO-SDS gel (CBS Scientific) and separated at 150 V for 1.5 h. Protein was transferred to Amersham™ Protran™ 0.2 μm NC membrane (GE Healthcare Life Sciences) using a G2 Fast Blotter (ThermoFisher Scientific). Membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 and probed with anti-TRPV4 (Alomone) and with anti-ß-actin (Cell Signaling) as a loading control. Appropriate HRP-conjugated secondary antibodies were used and the membrane developed with Super Signal Femto ECL (ThermoFisher Scientific) and imaged with a ChemiDoc-IT2 (link) bioimaging system (UVP).
Protocol detail
Western Blot Analysis of TRPV4 in Lung
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Actin
Adult
Antibodies
Assay
Buffer
Cultured medium
Detergent
Epithelial cells
Inhibitors
Isolation
Lung
Membrane
Milk
Mouse
Phosphatase
Protease
Protein
Ripa
Saline
Trpv4
Tween 20
Corresponding Organization :
Other organizations : University of Delaware
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Variable analysis
independent variables
- Isolation of lungs
- TRPV4 expression
- Culturing of adult mouse lung epithelial cells (MLE12) in HITES medium with 1% PS
- Lysis of MLE12 cells in RIPA with 2X HALT protease and phosphatase inhibitors
- Protein homogenization and quantification using a detergent compatible Lowry assay
- Denaturation of protein at 95°C in LDS sample buffer containing DTT
- Protein loading at 20 μg per lane into a 4–12% TEO-SDS gel
- Protein separation at 150 V for 1.5 h
- Protein transfer to Amersham™ Protran™ 0.2 μm NC membrane using a G2 Fast Blotter
- Membrane blocking with 5% milk in Tris-buffered saline with 0.1% Tween 20
- Probing with anti-TRPV4 and anti-ß-actin antibodies
- Use of appropriate HRP-conjugated secondary antibodies
- Membrane development with Super Signal Femto ECL and imaging with a ChemiDoc-IT2 bioimaging system
- Adult mouse lung epithelial cells (MLE12) for TRPV4 expression
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