Protease inhibitor cocktail

To express TIAM-1 N-terminal domain and UNC-119 for Co-IP assays, Human Embryonic Kidney Cells 293 (HEK293T) were cultured with a standard protocol in DMEM supplemented with 10% FBS. 2 x 10⁵ cells/well (in a 6-well dish) were transfected with the expression plasmid containing 1.5 μg TIAM-1N539 and 1 μg 3xFLAG-UNC-119-MYC using HyFectin transfection reagent kit. Two days after transfection, cells were lysed in 250 μl of lysis buffer (50mM Tris-Base, 150mM NaCl, 1% IGEPAL CA-630, pH 7.4, protease inhibitor cocktail (MedChemExpress)), and the lysates were then sonicated for 10 minutes for fully lysis. The resulting lysates were centrifuged at 16,200 ×g for 10 minutes at 4°C, and the supernatants were incubated with 20 μl of anti-FLAG M2 Affinity Gel (Sigma-Aldrich) at 4°C with agitation overnight. After washing three times with the lysis buffer, the samples were eluted with SDS loading buffer and were boiled for 3 min. SDS-PAGE and western blot were then performed using standard protocols. Anti-HA (Proteintech) and anti-FLAG M2 (Sigma-Aldrich) mouse antibodies were used at 1:2000 dilutions and 1:1000 dilutions, respectively, and HRP-conjugated goat antibodies to mouse were used at 1:10,000 dilutions (Proteintech). Details for plasmids used in co-IP assays can be found in S1 Table.

Cells were washed twice with ice-cold phosphate buffer saline, harvested, and resuspended in RIPA Lysis Buffer (Beyotime, Shanghai, China) plus protease inhibitor cocktail (MedChemExpress). Cell lysates were obtained by centrifugation at 12 000 r·min⁻¹ for 10 min at 4 °C. Equal amounts of each sample were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and then incubated with the designated antibodies. Antibody-specific labeling was revealed by incubation with HRP-conjugated secondary antibodies for 1 h and visualized with an electrochemiluminescence (ECL) kit (Millipore). Images were captured and analyzed with an ImageQuant Las 4000mini imaging system (GE Healthcare Life Science, Chicago, IL, USA). All immunoblotting assays were conducted with three biological replicates, and representative images are shown.

First, 293 T cells were seeded at 1 – 2 × 10⁷ cells per 10 cm dish and cultured overnight. After transfection with PEI for 48 h, the cells were harvested and washed with cold PBS following experimental treatments. Then, the cells were lysed with EBC buffer (50 mmol^.L⁻¹ Tris, pH 7.5, 120 mmol^.L⁻¹ NaCl and 0.5% NP-40) containing protease inhibitor cocktail (HY-K0010, 1:100, MedChem Express). After ultrasonication (power: 25%, sonicate 5 s, stop 5 s, five times), lysates were subjected to IP with anti-Flag beads (M2, Sigma) at 4 °C for 4–6 h or overnight, followed by washing in lysis buffer, SDS–PAGE electrophoresis and immunoblotting with the indicated antibody.

HEK-293T or HeLa cells were transfected with plasmids, as indicated in the figures, and cultured for 24 hours before collecting the protein lysate. All cells were washed with precooled PBS before collection and lysed in cell lysis buffer for western blotting and IP (Beyotime, P0013) supplemented with a 1% protease inhibitor cocktail (MedChemExpress, HY-K0010) and a 1% phosphatase inhibitor cocktail (Yeasen, 20109ES05) for up to 30 min at 4°C, after which they were centrifuged at 12,000 g for 10 min. Rat cerebral cortex and spleen samples were homogenized in cell lysis buffer with an electric homogenizer, lysed for 1 h at 4°C, and centrifuged at 12,000 g for 10 min at 4°C. The collected supernatants were used for IP using a previously described protocol 40 (link).
The antibodies used for IP were as follows: anti-eIF2α (Proteintech, 11170-1-AP), anti-phospho-eIF2α (Ser51) (Cell Signaling Technology, 3398), and anti-DDDDK (Flag)-tag (Abclonal, AE092).

MDBK cells were infected with BoHV-1 and harvested at the indicated time points. The cells were lysed with the cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, 0.1% SDS, 5 mM sodium orthovanadate) supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and the protease inhibitor cocktail (MedChem Express, Princeton, NJ, USA). Briefly, about 20 to 30 µg of the cell lysates was used to separate proteins on 10% SDS-PAGE, and the proteins isolated on the gel were transferred to PVDF membranes, which were then blocked with 5% nonfat dry milk in Tris-Buffered Saline Tween-20 (TBST, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). The membranes were incubated with appropriate primary antibodies diluted in TBST supplemented with 5% nonfat dry milk overnight at 4 °C, and then washed five times with TBST. Finally, the incubated membranes were treated with a horseradish peroxidase-conjugated secondary antibody for 2 h as previously described [48 (link),49 (link),50 (link)]. Protein bands were quantified and analyzed by densitometry using AlphaView software (version 3.4; ProteinSimple, Santa Clara, CA, USA).

Cells were lysed with RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing phosphatase inhibitor and protease inhibitor cocktail (Med Chem Express, Monmouth Junction, NJ, USA). Total protein was separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dried milk for 1 h at room temperature and incubated with the indicated primary antibody overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. The protein bands were visualized with ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). All antibody information is shown in Supplementary Table 1.