Anti mouse irdye conjugated secondary antibodies

Total proteins from tissues were extracted by homogenizing in RIPA buffer premixed with protease inhibitor cocktail (Sigma, St. Louis, MO). Proteins concentrations were determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL). Total protein (50 μg) was separated on 12% mini PROTEAN polyacrylamide gels and then transferred to polyvinylidenefluoride (PVDF) (Life Technologies Carlsbad, CA) using iBlot gel transfer system. Membrane was blocked using Odyssey blocking buffer for 1 h at room temperature. Membranes were incubated overnight with rabbit polyclonal to Gli-1 (SC-20687), rabbit polyclonal to Shh (SC-h160), goat polyclonal to β-actin (SC-1616) (1:1000) (Santa Cruz Biotech., Dallas, TX), and mouse monoclonal antibodies against KRAS (ab-55391) (1:1000) (Abcam, Cambridge, MA). After washing with TBST buffer, the membrane was further incubated with their corresponding antirabbit, antigoat, and antimouse IR dye conjugated secondary antibodies (1:10000) (LI-COR Biosciences, Lincoln, NE) for 60 min and visualized using LI-COR imaging system. Expression levels of desired protein were normalized against β-actin (SC-1616) protein expression levels.