Tissue lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The lysate was adjusted to 3.75 mg/ml, and then mixed with 5X Sample Buffer to become final 3 mg/ml in 1X Sample Buffer. The lysate was heated at 95°C for 5 min, and cooled rapidly.