Protease and phosphatase inhibitor

To quantify protein levels, western blotting was carried out as previously described (Autry et al., 2011 (link); Gideons et al., 2014 (link); Suzuki et al., 2017 (link)). To measure protein level in cultured neurons, cultured hippocampal neurons were lysed in Laemmli sample buffer containing phosphatase and protease inhibitors (Roche). To quantify protein level in hippocampus, hippocampus was dissected and lysed using RIPA buffer [50 mM Tris, pH 7.4, 1% Igepal® CA-630, 0.1% SDS, 0.5% sodium deoxycholate, 4 mM EDTA, 150 mM NaCl, phosphatase and protease inhibitors (Roche)], and lysate was mixed with Laemmli sample buffer. Samples were boiled for 5 min at 95°C. Then samples were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. Primary antibodies were incubated with membrane at following dilutions: 1:500 for anti-eEF2K (Abcam, ab45168), 1:5,000 for anti-phosphorylated eEF2 (Thr56) (Cell Signaling, 2331), 1:2,000 for anti-total eEF2 (Cell Signaling, 2332), 1:1,500 for anti-BDNF (Abcam, ab108319), 1:20,000 for anti-ERK1/2 (Cell Signaling, 4695), 1:20,000 for anti-phospho-ERK1/2 (Cell Signaling, 4370) and 1:50,000 for anti-GAPDH (Cell Signaling, 2118). Membranes were incubated with HRP anti-rabbit secondary antibody (Vector Laboratories, Inc.). Chemiluminescence was detected using Clarity Western ECL substrate (Bio-Rad). Band intensities were analyzed by ImageJ

For kinase assay in vitro, 12 μg CDK7-Flag and YAP-Flag plasmids were transfected into HEK293T cells and incubated for 60 h, respectively. Collected these cells and added 1 ml 1% NP-40 lysis buffer (Beyotime) containing protease and phosphatase inhibitor (Roche). Then obtained the supernatant and added 30 μl Protein A/G beads (NCM Biotech) to incubate at 4 °C overnight. The next day, added 5 μg anti-Flag antibody to each reaction and incubated at 4 °C for 4 h. Washed the beads with 500 μl 1 × kinase reaction buffer (25 mM Tris pH 7.5, 2 mM DTT, 10 mM MgCl₂, 200 μM ATP) containing protease and phosphatase inhibitor (Roche) for three times. Then added 10 μg YAP-beads and 0.5 μg CDK7-beads to 100 μl reaction system and incubated at 30 °C water bath for 30–60 min. Boiled water bath for 10 min was used to terminate the reaction and then froze with liquid nitrogen. Subsequent mass spectrometric identification and data analysis were completed by SpecAlly Life (Wuhan) Science and Technology Co. Ltd.

PAM were seeded in p60 plates (6 × 10⁶ cells/plate) and mock infected or infected with NH/P68 or Arm/07/CBM/c2 (2 PFU/cell) for 7 or 15 h. Cells were then mock treated or treated with IFN-I (250 U/ml) for 1 h. The whole cell extract (WCE), cytoplasmic fraction and chromatin fraction were isolated as described previously (Méndez and Stillman, 2000 (link); García-Belmonte et al., 2019 (link)). Briefly, cells were resuspended in a buffer composed by 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors (Roche). This lysate corresponded to the WCE. Then, Triton X-100 was added followed by incubation for 5 min on ice. The nuclei were then centrifuged for 4 min at 3,600 rpm at 4°C. The supernatant was collected and centrifuged at 14,000 rpm for 15 min at 4°C corresponding to the cytoplasmic fraction. The pellet obtained in the first centrifugation was washed with the previous buffer and resuspended in a buffer containing 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 0.1 mM PMSF and protease and phosphatase inhibitors (Roche). After incubation on ice for 30 min, the pellet lysate was centrifuged for 4 min at 4,000 rpm at 4°C. The pellet corresponding to the chromatin fraction was washed and resuspended in loading buffer.

For Western blotting, total cell lysates were obtained using NP40 buffer (0.1% Nonidet P-40 (NP-40), 0.4 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). For the isolation of cytosolic fractions, cells were lyzed in buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.15% NP-40, 1 mM DTT) with protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 12,000 g for 30 seconds at 4°C, and supernatants were taken as cytosolic fraction. The pellets were washed with ice-cold phosphate-buffered saline (PBS) and lyzed in buffer B (20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 1 mM DTT). After sonication (5 seconds at 50 watt), samples were centrifuged at 12,000 g for 15 minutes at 4°C, and supernatants were taken as nuclear fractions. Protein concentration was determined using Bradford assay (Biorad). Thirty micrograms of protein was separated using SDS-PAGE and transferred to Amersham Hybond-P PVDF Transfer Membrane (GE Healthcare; RPN303F). Membranes were blocked in 5% milk in Tris-buffered saline-0.01% Tween20 and incubated with the indicated antibodies. Membranes were visualized using a ChemiDoc XRS+ System (Bio-Rad) using Image Lab software version 5.2.1 (Bio-rad). Images have been cropped for presentation, but full-size images are presented in S4 Fig.