Rabbit anti β actin

After ALV-J infected DF-1 cells for 48 h, the cells were collected, and then the cells were lysed with RIPA and the supernatant was collected. The protein concentration was then measured using a BCA protein concentration assay kit (Beyotime, Shanghai, China), and finally, a western blot analysis was performed.
A 12% sodium dodecyl sulfate-polyacrylamide (SDS) gel (GenScript, Nanjing, China) was used for protein separation. Then, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Polyvinylidene Fluoride) following 1-h blocking with 5% defatted milk. Then, primary antibodies were added and incubated at 4 °C overnight. After washing with tris-buffered saline tween (TBST), secondary antibodies were added for 1-h incubation at RT. Primary antibodies, including Mouse anti-gp85, JE9, donated by Professor Aijian Qin of Yangzhou University, and Rabbit anti-β-Actin were purchased from Novus Biologicals (Englewood, CO, USA). Secondary antibodies, including HRP Goat Anti-Mouse IgG(H+L), purchased from ABclonal (Wuhan, China), and Goat pAb to Rb IgG (HRP), purchased from Abcam (Cambridge, UK).

Cells were collected after 48 h of transfection and lysed using Radioimmunoprecipitation lysis buffer consisting of 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate and protease inhibitors. The samples were denatured at 100C for 10 min before loading onto 10% SDS-polyacrylamide gels. Separated proteins were then transferred onto nitrocellulose membranes and blocked with 5% non-fat milk in PBS with 0.1% Tween-20 for overnight. Membranes were then incubated overnight with 1/1000 rabbit anti-CDK12 (Novus Biologicals, Abingdon, United Kingdom) and 1/1000 rabbit anti-β-actin (Novus Biologicals, Abingdon, United Kingdom). After three washes, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H + L) (Novus Biologicals, Abingdon, United Kingdom) for 2 h at room temperature. Signals were revealed by autograph using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, United States).

Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [26 (link)] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [19 (link)] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology). Probes used for immunofluorescence: Alexa 633-conjugated concanavalin A (ConA) (Thermo Scientific). Antibodies used for flow cytometry: mouse anti-MHC I W6/32 (Novus Biologicals); mouse anti-NGFR (Sigma-Aldrich); and Alexa 633-conjugated goat anti-mouse IgG (Thermo Scientific).