Irdye 680 and 800 secondary antibodies

S-Nitrosoglutathione (GSNO, N4148), DTT (D0632), and Puromycin (P8833) were from Sigma (St. Louis, MO). L-NAME HCl (S2877) and Cycloheximide (CHX, S7418) were purchased from Selleck Chemicals (Houston, TX). Cavosonstat (N91115) were from STA for Nivalis (Shanghai). TCA Protein Precipitation Kit was purchased from Sangon Biotech (Shanghai, China). Antibodies against VEGFD(C-12), FLK1 (A-1), PECAM-1 (H-3), and c-Myc were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against VEGF receptor 3 (VEGFR3) were from Cell Signaling Technology (Danvers, MA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), HA, VEGFD, and LYVE1 were from HUABIO (Hangzhou, China). The IRDye 680 and 800 secondary antibodies were from LI-COR Bioscience (Lincoln, Nebraska). Alexa555- and Alexa488-conjugated antibodies were from Molecular Probes (Eugene, OR).

Frozen mouse liver samples were homogenized in the presence of lysis buffer (15% w/v, Invitrogen) containing protease inhibitors (Sigma-Aldrich), 1mM Na₃VO₄, and 50mM NaF, clarified by centrifugation twice at 12,000 g for 30 min at 4°C, and stored at -80°C. The samples were subjected to SDS-PAGE and immunoblotting using standard methods. Blots were blocked with Odyssey blocking buffer (LI-COR Bioscience) and probed with the following primary antibodies: IRF1 (8478, 1:500, Cell Signaling), IRF3 (4302, 1:400, Cell Signaling), phospho-IRF3 (Ser396) (4947, 1:200, Cell Signaling), IRF5 (20261S, 1:200, Cell Signaling), β-actin (3700, 1:5000, Cell Signaling). IRDye 680 and 800 secondary antibodies (LI-COR Bioscience) were used to visualize protein bands with an Odyssey two-color detection imaging system (LI-COR, Bioscience).

Total protein of rNP cells seeded in a 6 cm dish was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% protease inhibitors for 30 min at 4°C. The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). An equal amount of protein (30 μg) was separated by SDS-PAGE and transferred onto nitrocellulose filter membranes (Millipore, Billerica, City, State, United States). After blocking with 5% non-fat milk for 1 h, membranes were respectively incubated with primary antibodies against p53 (1:1,000), p21 (1:500), p-Mst1/2 (1:1,000), Mst1/2 (1:1,000), p-Lats1/2 (1:1,000), Lats1/2 (1:1,000), p-Yap (1:1,000), Yap (1:1,000), Taz (1:1,000), GAPDH (1:1,000) at 4°C overnight. Then, IRDye 680 and 800 secondary antibodies (LI-COR, Lincoln, NE, United States) were added and incubated for 1 h. The signals were visualized with Odyssey Infrared Imaging System (LI-COR). Immunoreactive bands were quantified by Quantity ONE software (Bio-Rad, Hercules, CA, United States). GAPDH was used as the internal standard of total target proteins, and phosphorylated proteins were normalized to their corresponding total protein.