Phosphatase inhibitor

Total protein was extracted from human tissues and cell lines using protein extraction buffer containing a 1% protease inhibitor cocktail (Targetmol, Shanghai, China) and a phosphatase inhibitor (Cat. No. 4906845001, Roche, Basel, Switzerland). The lysate was centrifuged at 4°C at 14,000 rpm for 15 min, and proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Massachusetts, USA). After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated at 4°C overnight with primary KLF4 (ab215036, Abcam, San Francisco, USA), p-JNK (sc-293136, Santa, California, USA), and JNK (sc-7345, Santa, California, USA) antibodies and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (TA-08, ZSGB, Beijing, China) antibodies. After washing with Tris-buffered saline with 1% Tween-20 (TBST), the membranes were incubated with HRP-conjugated secondary antibodies (ZSGB-Bio, Beijing, China) for 1 h at room temperature. The blots were visualized using enhanced chemiluminescence reagents (ECL) detection (Amersham Biosciences Fairfield, CT, USA).

Twelve C¹-, C³-, or C⁶-modified β-carboline derivatives were synthesized as previously described [27 (link),28 (link)]. The structures of these β-carboline derivatives are summarized in Table 1.
Dynasore, U0126, LY294002, and ribavirin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO) for in vitro studies. The protease inhibitor cocktail and the phosphatase inhibitor were obtained from TargetMol (Boston, MA, USA). Antibodies against β-actin, p-ERK, ERK, p-Akt, Akt, goat anti-rabbit IgG, and goat anti-mouse IgG were purchased from Cell Signaling Technology (Topsfield, MA, USA). The mAb against NDV nucleoprotein was prepared in our laboratory.
DF-1, Hela, Vero, and BHK-21 cells originally obtained from ATCC (Manassas, VA, USA) were purchased from the Cell Bank of Chinese Academy Sciences (Shanghai, China). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (Gibco, Amarillo, TX, USA) at 37 °C with 5% CO₂.
Four NDV strains, including F48E9, Blackbird/China/08, PPMV-1/SX-01/Ch/15, and La Sota were provided by the College of Veterinary Medicine, Northwest A&F University (Xianyang, China). These viruses were plaque-purified three times. Viruses were propagated in 9-day-old to 11-day-old SPF chicken embryos, titrated by plaque assay, and stored at –80 °C.