Ab38611

To explore the protein expression in ACTH-secreting pituitary adenomas, proteins were precipitated from phenol-ethanol supernatant after RNA isolation according to the manufacturer’s protocol. Equal amounts (20 µg) of protein were separated using a SDS-polyacrylamide gel, transferred onto nitrocellulose membrane (NC) and incubated in blocking solution (TBS buffer containing 5% non-fat dry milk) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibody (anti-SPP1, epitomic #2671-1, 1:500; anti-COL1A1, epitomic, #6690-1, 1:500; anti-NT5E, epitomic, #5362-1, 1:1000; anti-HTRA1, Abcam, ab38611, 1:200; anti-ANGPT1, Abcam, ab8451,1:200; anti-GAPDH:1:2000, epitomic, #5632-1) and subsequently with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Immunocomplexes were visualised using the ECL (electrochemiluminescence) Western Blotting Detection Reagents (Millipore, ‎Billerica, MA, USA) and detected via a Kodak film exposure detection system. Quantification of the bands was performed using the Quantity-One software (Bio-Rad, ‎Hercules, CA, USA).