Prime ecl detection systems

Cells were lysed in Laemmli sample buffer, boiled for 5 min, sonicated for 15 s and spun down. Lysates were fractionated using 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF filters (0.45 µm, Immobilon). ECL or Prime ECL detection Systems (GE Healthcare) with HRP-conjugated secondary antibodies (GE Healthcare) were used for detection. Bands were quantified using Image J. Levels of phospho-proteins were calculated after correction to total levels of the relevant protein. Antibodies: pThr18/Ser19-MLC2 (1:750, #3674), MLC2 (1:750, #3672) and RhoA (1:1000, #2117) from Cell Signalling Technology; GFP (1:10000, sc-8334) from Santa Cruz Biotechnology; GAPDH (1:10000, MAB374) from Millipore.

Cells were lysed in Laemmli buffer and snap frozen. Lysates were then boiled, sonicated for 15 s and spun down. Cell lysates were fractionated using sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels in non-reducing conditions, and transferred subsequently to PVDF filters. Membranes were blocked in 5% BSA in 0.1% Tween 20-TBS. Primary antibodies were incubated overnight at 4°C. For detection, ECL or Prime ECL detection systems coupled to HRP-conjugated secondary antibodies (GE Healthcare) with X-ray films and an Amersham Imager 600 were used. Bands were quantified using ImageJ. Levels of phospho-proteins were calculated after correction to total levels of the relevant protein.