Horseradish peroxidase conjugated igg

We used commercial rat and mouse antibodies: anti-GFP (Nacalai), anti-HA (Roche Diagnostics, Cell Signaling), anti-Flotillin-1, anti-Caveolin-1, anti-Rac1, anti-GM130 (BD), horseradish peroxidase-conjugated IgG (Amersham Biosciences), and Alexa 488-, Alexa 555-, and Alexa 594-conjugated IgG (Molecular Probes).

Proteins (20 ug/sample) were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blotted at 4 °C overnight with primary antibodies. The membranes were washed five times with 10-mM Tris–HCl (pH 7.5) plus 150-mM NaCl (Tris buffered saline, TBS) that contained 0.2% Tween-20, and incubated with horseradish peroxidase-conjugated IgG. After the removal of excess antibodies by washing with TTBS, specific binding was detected using a chemiluminescence detection system (Amersham, Berkshire, UK) according to the manufacturer's instructions. Mouse monoclonal Ab to IGF-II, phospho-ERK, SUMO1 and rabbit polyclonal Abs to SUMO2/3 was from Santa Cruz Biotechnology. Rabbit polyclonal Abs to phospho-p65, phospho-src, phospho-STAT3 and mAbs to ubiquitin were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal Ab to E-cadherin was from BD science. Rabbit polyclonal Abs to GAPDH were from Lab Frontier (Seoul, Korea), and horseradish peroxidase conjugated goat Abs to mouse or rabbit IgG were from Amersham and Sigma (Sigma-Aldrich, MO, USA).