Protease inhibitor cocktail

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).

Cells were lysed in lysis buffer supplemented with protease inhibitor cocktail (TargetMol, USA) at 4 °C for 20 min. The western blotting assay was performed as described previously [21 (link)].

Cells were resuspended in RIPA lysis buffer mixed with an EDTA‐free protease inhibitor cocktail (#C0001, TargetMol, MA, USA). The prECLeared lysate was incubated with magnetic beads (Thermo Fisher Scientific, Carlsbad, CA, USA) and then immunoprecipitated overnight at 4 °C using the indicated antibodies. The protein complex was then subjected to western blotting. Briefly, the samples were separated using sodium dodecyl‐sulfate polyacrylamide gel electrophoresis and then blotted on PVDF membranes (Millipore, Billerica, MA, USA). The membrane was then blocked for 30 min with 5% nonfat milk, and incubated overnight with primary antibodies. This was followed by incubation with horseradish peroxidase‐conjugated secondary antibodies. The signal was then visualized using ECL (WBKLS0500, Millipore). GAPDH was used as the loading control. The antibodies in the study were listed in Table S5 (Supporting Information).

Hepa 1-6 cells reached confluence in 6-well plates and were then incubated with different concentrations of NTP-217 for 4 h. Next, the cells were washed twice with cold PBS and then lysed using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) with phosphatase inhibitor cocktail I (cat. no. C0002; TargetMol) and Protease Inhibitor Cocktail (cat. no. C0001; TargetMol). Protein concentration was measured with BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Inc.). Samples (30 μg/lane) were subjected to 12% SDS-PAGE and then transferred to PVDF membranes (Millipore Sigma). The membranes were blocked with QuickBlock™ Blocking Buffer for Western Blot (Beyotime Institute of Biotechnology) and then incubated overnight at 4˚C with primary antibodies against Poly [ADP-ribose] polymerase 1 (1:5,000; cat. no. 66520-1; ProteinTech Group, Inc.), caspase-3 (1:10,000; cat. no. ab32499; Abcam), cleaved caspase-3 (1:1,000, cat. no. 9661, Cell Signaling Technology, Inc.) and GAPDH (1:10,000; cat. no. abs132004; Absin). The membranes were washed with TBS with 0.05% Tween-20 (TBST) buffer thrice for 5 min each and incubated with a secondary antibody (1:10,000; cat. no. abs20002; Absin) for 1 h at room temperature. Protein bands were visualized by using an ECL reagent (Thermo Fisher Scientific, Inc.) and detected with ECL Western Blotting Detection System (Bio-Rad Laboratories, Inc.).

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.