Mrckα

Tissue fragments and cultured cells were lysed with Laemmli buffer. Lysates were separated by a 10% SDS-PAGE and Western blotting was carried out following standard protocols. For detection, the following primary antibodies were used: MRCKα (#374568), MRCKβ (#374597), GAPDH (#25778; all SantaCruz Biotechnology, Dallas, TX, USA), pMLC (#3674), pCofilin (#3311; all Cell Signaling, Danvers, MA, USA), β actin (#AB6276, Abcam, Cambridge, MA, USA). For detection, appropriate HRP coupled secondary antibodies were used: horse-anti-mouse IgG (#VECTPI2000), goat-anti-rabbit IgG (#VECTPI1000, all Vector Laboratories, Burlingame, CA, USA). LuminataTM Western HRP Chemiluminescence Substrates detection reagent (Milipore, Hellerup, Denmark) was used for chemiluminescence, which was then detected with Medical X-ray film (AGFA, Birkerød, Denmark). The intensity of the bands was quantified using ImageJ software.

Western blot was performed as previously described^{18 (link)}. Briefly, half of the left lobe of mouse lungs were homogenized in 300 μl of lysis buffer (1× Reporter lysis buffer (Promega, Madison, WI, USA), containing protease inhibitor (Roche, Basel, Switzerland)) using a TissueLyser II (Qiagen, Germantown, MD, USA). Supernatants were used for SDS-PAGE and western blots. Thirty micrograms of total protein were loaded on 10% SDS-PAGE gels, transferred onto PVDF membrane and probed with primary antibodies against DDK tag (Origene, Rockville, MD, USA), ZO-1, occludin, and VE-cadherin (Invitrogen, Rockford, IL, USA), β1-Na⁺, K⁺-ATPase (Millipore, Billerica, MA, USA), MRCKα (Santa Cruz, Dallas, TX, USA) and glyceraldehyde-3-phosphate dehydrogenase GAPDH (EMD Millipore, Burlington, MA, USA). Data were analyzed using the NIH Image J software.