Hrp conjugated donkey anti rabbit igg

Testes from 10-week-old mice were homogenized in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X–100, 5 mM EDTA and 1 mM Na₃VO₄) containing protease inhibitors (Roche). Western blot was carried out as described previously (Jiang et al., 2014 ). The primary antibodies are rabbit anti-β-actin (Abcam, ab8227; 1:3,000), rabbit anti-UBL4A (Yang et al., 2007 (link)) and rabbit anti-UBL4B (Yang et al., 2007 (link)). The secondary antibody is HRP-conjugated donkey anti-rabbit IgG (BioLegend, 406401; 1:10,000).

Fibroblasts of proband A-4 and two healthy donors were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 g/ml streptomycin. Near-confluent fibroblasts were treated with STO-609 (5 µM; Merck) or vehicle (DMSO). Two hours later, lysates were prepared with lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, pH 8, protease inhibitors [cOmplete Mini, Merck]), followed by agitation and centrifugation. Supernatants were subjected to SDS-PAGE and semidry immunoblotting. Primary antibodies used (all rabbit, 1:1000) were anti-CaMKIV (ThermoFisher PA1-542), anti-CREB (Cell Signaling 9197), and anti-phospho-CREB (Cell Signaling 9198). Secondary antibody was HRP-conjugated donkey anti-rabbit IgG (BioLegend 406401). Chemiluminescent detection was performed with ECL Prime (Amersham). Protein abundance was quantified by signal density (ImageJ).

Virus-infected or plasmid-transfected cells were harvested and lysed with RIPA lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol, supplemented with a protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific, MA, United States). The mixture was centrifuged at 12,000 g, 4°C for 10 min and the resulting supernatants were mixed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled for 5 min. Proteins from the cell lysates were separated using SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad Laboratories, CA, United States). The membrane was blocked with 5% skim milk prior to incubation with the indicated primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Biolegend, CA, United States) or (HRP)-conjugated donkey anti-rabbit IgG (Biolegend, CA, United States) were used as secondary antibodies. Primary antibodies used in this study included mouse-anti-Myc (Thermo Fisher Scientific, MA, United States), rabbit-anti-FLAG (ab1162, abcam, Cambridge, United Kingdom), rabbit-anti-HA (ab9110, abcam, Cambridge, United Kingdom), and mouse-anti-PEDV-N (SD 6–29, Medgene labs, SD, United States), rabbit polyclonal anti-Zinc finger antiviral protein (ab154680, abcam, Cambridge, United kingdom).