Ab134309

For tissue samples, about 0.2 g of tissue was grinded into powder in liquid nitrogen. Protein extracts of frozen tissue powder and cells were prepared by using RIPA buffer in the presence of proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA). Total protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Bredford, USA). Anti-REG3A (Abcam, Ab134309), anti-MCM2 (Abcam, Ab4461), anti-MCM3 (Abcam, Ab128923), anti-PRAP1 (Abcam, Ab118186), anti-PRAP2 (Abcam, Ab176330), anti-pAKT(Cell Signaling Technology (CST), #4060, Danvers, MA, USA), anti-AKT (CST, #4691), anti-pERK1/2 (CST, #9101S), anti-ERK1/2 (CST, #9102S) and anti-GAPDH (CST, #5174) were used in Western analysis in accordance with the manufacturer's instruction. Signals were visualized with enhanced chemiluminescenct substrate (ECL, BioRad, Richmond, CA, USA) by exposure to films.

Cell lysates were reacted with Anti-REG3A (Ab134309, Abcam) or control IgG (Abcam) for 1 h at 4°C, then with protein A-agarose (150 μg protein A) for 3 h at 4°C. Precipitates were washed three times in lysis buffer and detected by western blot analysis.