Irdye 680lt goat anti mouse lgg

Proteins were extracted from the transfected cells using a lysis buffer (Upstate Biotechnology, Lake Placid, NY, USA). The protein level was determined using protein assay reagents following the standard protocols (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, 25 µg of total protein was loaded on 12% PAGE gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories). Following the transfer, the membranes were blocked with Odyssey^® blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), and then they were incubated in primary antibodies buffer (primary antibodies, Odyssey Blocking Buffer, 0.1% Tween^® 20) overnight at 4°C. Two primary antibodies, anti-phosphatase-and-tensin homolog (PTEN) antibody and anti-SMAD4 antibody, were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The following day, the membranes were washed four times for 5 minutes in Tris-buffered saline and Tween 20 prior to being incubated in the secondary antibodies IRDye^® 680LT Goat anti-Mouse lgG or IRDye 680LT Goat anti-Rabbit lgG (LI-COR) plus Odyssey blocking buffer and 0.1% Tween 20 at 1:20,000 dilution for 1 hour. Finally, the membranes were visualized on an Odyssey CLx imaging system, model:Ody-3086 (LI-COR).