Anti clathrin heavy chain

Wortmannin, dynasore, SU6655, NH₄Cl, SP600125, IPA-3, MβCD and chlorpromazine were obtained from Sigma (Sigma, MO, United States), Akti-1/2 and bafilomycin A1 were obtained from Abcam (Abcam, Cambridge, United Kingdom), nystatin and EIPA was obtained from Solarbio (Solarbio, Beijing, China).
The rabbit anti-dynamin-2, anti-clathrin heavy chain, anti-Rab5, anti-Rab7, anti-PI3K p85 (phos pho Y458) + PI3 Kinase p55 (phospho Y199), anti-Akt, anti-Src, anti-Src (phospho Y416), anti-phospho-Akt (Ser473) and anti-GAPDH monoclonal antibodies were obtained from Abcam (Abcam, Cambridge, United Kingdom). The rabbit anti-JNK and anti-JNK (Thr183/Tyr185) monoclonal antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, Danvers, United States). The rabbit anti-PI3 Kinase p85 alpha polyclonal antibody, the mouse anti-Flag monoclonal antibody, goat anti-rabbit and goat anti-mouse HRP-labeled secondary antibody, Alexa fuor-488-conjugated anti-mouse, Cy™3-conjugated anti-rabbit IgG (H + L) and mounting medium with DAPI was purchased from Abcam (Abcam, Cambridge, United Kingdom). The rabbit anti-caveolin-1 monoclonal antibody was obtained from Beyotime (Beyotime Biotechnology, Shanghai, China). The mouse anti-BRSV G protein monoclonal antibody was provided by China Animal Health and Epidemiology Center.

PAMs or Marc-145 cells that were grown in 12-well cell plates or in 15 mm covered, glass-bottomed petri dishes were washed with PBS and were fixed with 4% paraformaldehyde (Beyotime, P0099) for 10 min, and they were then permeabilized with 0.5% Triton X-100 (Beyotime, P0096) in PBS for 5 min. The cells were blocked in Immunol staining blocking buffer (Beyotime, P0260) for 30 min at room temperature. Subsequently, the primary antibodies were incubated overnight at 4°C. After washing to remove unbound antibodies, the cells were incubated with the appropriate Alexa Fluor 488 or 555 conjugated secondary antibodies at room temperature for 1 h in the dark. Washing three times again, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, C0065) for 5 min. Finally, the fluorescence images were taken using an inverted fluorescence microscope (NIKON ECLIPSE Ti2-U) or a confocal laser scanning microscope (TCS-SP5, LEICA). Antibodies used in the immunofluorescence and confocal microscopy: anti-Myc antibody (1:800; Cell Signaling Technology, 2276s), anti-PRRSV N antibody (4A5) (1:1500; Jeno Biotech, 9041), anti-clathrin heavy chain (1:1000, Abcam, ab21679), anti-Mouse IgG Alexa Fluor 488 (1:1000, Cell Signaling Technology, 4408s), and anti-Rabbit IgG Alexa Fluor 555 (1:1000, Cell Signaling Technology, 4413s).

SUM-159 cells plated on coverslip chambers (Thermo Fisher, Cat. # 155379) were fused at their corresponding time points, fixed with 4% PFA (EMS), and then permeabilized and blocked with blocking solution (0.5% Triton X-100, 10% BSA in PBS) for 1 h. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C. After 3 washes (10 min) with PBS, cells were incubated with Alexa Fluor-conjugated secondary antibodies. The following primary antibodies were used: Anti-Yap1 (Cell Signaling 14074S, 1:250), Anti-p21 (Cell Signaling, Cat. # 2947S, 1:200), Anti-pH3 (anti-Phospho-Histone H3 (Ser10); Cell Signaling, Cat. # 3377, 1:200), Anti-clathrin heavy chain (Abcam, Cat. # ab21679, 1:200), Anti-AP-2 (Abcam, Cat. # ab189995, 1:200), Anti-Glut1 (Abcam, Cat. # ab40084, 1:100), Anti-CD98 (BioLegend, Cat. # 315602, 1:200) and Anti-CD147 (BioLegend, Cat. # 306202, 1:200). For imaging and quantification, at least a total of 15 fields of view were randomly chosen by Hoechst 33342 nuclear staining (Thermo Fisher, Cat. # 62249) and imaged by Zeiss LSM880 confocal or NIKON TIRF microscope. At least three different samples were quantified per treatment type at each respective time point.