Hrp conjugated goat anti mouse

Western blotting was carried out as described previously (Lin et al., 2019 (link); Wu et al., 2019 (link)). Briefly, the proteins from each cell layer extract were quantified using the BCA Protein kit (Beyotime Biotechnology, Shanghai, China). Protein extracts were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). Membranes were blocked with 5% non-fat milk in Tris–buffered saline for 1 h at room temperature. The membranes were incubated with primary antibodies, including anti-RAP2a (1:2,000; catalog no. NBP2-24574; Novusbio), DNMT3a (1:500; catalog no. 2160; Cell Signaling Technology), and anti-β-actin (1:3,000; catalog no. AP53385; Abgent) at 4°C overnight, followed by incubation with HRP-conjugated goat anti-rabbit (1:5,000; catalog no. sc-2004; Santa Cruz Biotechnology; RRID:AB_631746) or HRP-conjugated goat anti-mouse (1:5,000; sc-2005; Santa Cruz Biotechnology; RRID:AB_631746) secondary antibodies at 37°C for 1 h. The immunoreactive bands were visualized by using the ECL Plus Western Blot Detection kit (Amersham Biosciences United Kingdom), and densitometric quantification of band intensity from three independent experiments was carried out with the Image-Pro Plus 6.0 software. The relative expression level of the target protein was normalized to the intensity of the β-actin band.

Lipopolysaccaride (LPS) from E. Coli O111:B4 strain, Juglone, PiB, Phosphate Buffered Saline (PBS), Hanks' Balanced Salt Solution (HBSS), protease and phosphatase inhibitors were obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Dextran T500 and Ficoll was from GE healthcare (Orsay, France). Sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) and western blotting reagents were supplied by Bio-Rad (Hercules, CA, USA). The rabbit polyclonal antibodies against phospho-p47^phox sites (phospho-Ser345, phospho-Ser320, phospho-Ser304, phospho-Ser315, phospho-Ser328), p67^phox, and p47^phox were produced by our lab as described elsewhere (18 (link), 33 (link)). Anti-phospho(P)-ERK1/2, ERK1/2, P-p38, and p38 were from cell signaling Technology (Boston, MA, USA). HRP-conjugated goat anti-mouse were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Indirect immunofluorescence assays (IFAs) were performed on the 293T and DF1 cells. The monoclonal antibody JE9, which is specific to the Env of ALV-J, was used as the primary antibody19 (link). FITC-goat anti-mouse IgG was used as the secondary antibody. Western blot analyses were performed on cell lysates. JE9 or anti-β-actin or anti-chANXA2 antibody was used as the primary antibody, and HRP-conjugated goat anti-mouse or HRP-conjugated donkey anti-goat was used as the secondary antibody (Santa Cruz).

Human embryonic kidney (HEK293) cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum at 37°C, 5% CO₂ and transfected with 4 μg pRK5myc-hCB3_SH3− wild-type or R338W mutants using FuGENE (Roche). After 24 h, transfected cells were solubilized in a buffer containing Triton X-100 (Sigma-Aldrich), 1%; 150 mM NaCl; 50 mM Tris, pH 7.4, with protease inhibitor cocktail (Roche, Sussex, UK). Insoluble material was removed by centrifugation at 16, 100× g for 20 min. Phosphatidylinositol-3-phosphate (PI₃P/PtdIns-3-P) agarose beads (40 μl; Eschelon Biosciences) were incubated with cell lysates for 2 h at 4°C. Beads were washed four times in buffer. Proteins were eluted from beads by heating at 98°C for 3 min in 2 × sample loading buffer and then subjected to SDS-PAGE. Proteins binding to beads were detected by Western blotting using mouse anti-c-myc antibody (Sigma, 1:1000) and HRP-conjugated goat anti-mouse (Santa Cruz, 1:2000). Immunoreactivity was visualized using West Pico Chemiluminescent Substrate (Pierce). Expression levels of hCB3_SH3− and hCB3_SH3−R338W, and PI₃P pulldown assay results were assessed using an unpaired, two-tailed Student’s t-test.

For standard protein analysis protein lysates were separated on SDS-PAGEs and blotted on a Protan BA83 Nitrocellulose membrane (GE Healthcare). After saturating free binding sites with 5% milk powder in 1× TBS-T membrane was incubated with suitable primary antibodies overnight at 4 °C under constant agitation. After three times 5 min washing with TBS-T, membrane was incubated with matching HRP-coupled secondary antibodies (anti-mouse or anti-rabbit (Santa Cruze Biotechnology)) for 1 h at RT followed by another three washing steps. Signals were detected by chemiluminescence of HRP-coupled anti-mouse or anti-rabbit secondary antibodies (Santa Cruz Biotechnology) on a Gel Doc XR + Gel Documentation System (Biorad). Uncropped blots are provided in the Source Data file.
Used antibodies: Anti-CANX (1:1000, Abcam ref# ab31290), Anti-DHX36 (1:500, Santa Cruz ref# sc-377485), Anti-FLAG (1:2000, Sigma-Aldrich ref# F1804), Anti-FMR1 (1:2000, Linder et al. 2008), Anti-HA (1:2000, Covance ref# MMS-101R), Anti-HISTH2B (1:2000, Abcam ref# ab1790), Anti-PKR/EIF2AK2 (1:1000, Abcam ref# ab32052), Anti-PKR/EIF2AK2 (phospho T446) (1:1000, Abcam ref# ab32036), Anti-RPL22 (1:2000, Santa Cruz ref# sc-136413), Anti-TUBA4A (1:5000, Sigma-Aldrich ref# T5168), HRP-conjugated goat anti-mouse (1:5000, Santa Cruz ref# sc-2031), and HRP-conjugated goat anti-rabbit (1:5000, Santa Cruz ref# sc-2030)