Pa5 14117

NE-treated cells were fixed for 15 min using 4% paraformaldehyde and then blocked for 30 min using 5% donkey serum containing 0.3% Triton X-100. The cells were then incubated with the primary antibodies against CHGB (1 : 100; rabbit; PA5-52605, ThermoFisher Scientific) or SYP (1 : 100; rabbit; MA5-14532, ThermoFisher Scientific) for 3 and 1 h, respectively, followed by incubation with anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) secondary antibody for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.
For Adrβ2 and cytokeratin expression in tumor and adjacent normal tissues, the tissues were incubated with primary antibodies against Adrβ2 (1 : 100; rabbit; PA5-14117, ThermoFisher Scientific) and cytokeratin (1 : 100; mouse; MA1-82041; ThermoFisher Scientific) for 1 h at room temperature, followed by incubation with a cocktail of anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) and anti-mouse Alexa Fluor® 546 (1 : 100, A21045, ThermoFisher Scientific) for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.

Total protein from tumor and normal adjacent tissues were isolated using RIPA buffer (Thermo Scientific) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Thirty micrograms of proteins were then allowed to resolve in an SDS-polyacrylamide gel electrophoresis gel and the resolved proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was then incubated with the primary antibody against Adrβ2 (1 : 1000; rabbit; PA5-14117, ThermoFisher Scientific) for 1 h followed by goat anti-rabbit horseradish peroxidase (HRP) conjugate (1 : 3000; Biorad) and then developed using ECL reagent (Biorad). The membrane was also re-probed with β-actin antibody (1 : 2000; mouse; sc-47778, Santa Cruz Biotechnology) for 1 h followed by goat anti-mouse HRP conjugate (1 : 3000; 170-6516, Biorad) and then developed using ECL reagent. The blots were visualized and images captured, using a Geldoc (Biorad).