New clarity ecl substrate

Whole cell lysates were prepared using RIPA buffer containing 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA and 0.1% sodium dodecyl sulfate separated by electrophoresis in SDS-containing polyacrylamide gels (SDS-PAGE), and transferred onto poly-vinylidene difluoride (PVDF) membranes (Millipore, USA). The blots were incubated with the following antibodies against GFP, MEF2C (Santa Cruz Biotechnology, USA), and β-actin (Abcam, USA), then sequentially incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology, USA). Chemo-luminescent signals were visualized using NEW Clarity™ ECL substrate (Bio-Rad, USA).

Whole cell lysates were prepared using RIPA buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 0.1% SDS), separated by electrophoresis in SDS-containing polyacrylamide gels, and transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against DUSP6 and EGFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β-actin (Abcam, Cambridge, MA, USA) and then further incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Chemiluminescent signals were visualized using NEW Clarity ECL substrate (Bio-Rad, Hercules, CA, USA). For quantitative analysis, the density of the individual images was analyzed using ImageJ software (NIH, Bethesda, MD, USA).