Anti β tubulin mab

Whole cell lysates of PAMs or CRL-2843 cells were harvested in a 1× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as previously described [38 (link)]. Separated proteins were then transferred to PVDF membranes, as previously described [39 (link)]. Membranes were probed with homemade anti-sISG15 Mab-3D5E6 or rabbit anti-sISG15 polyclonal antibodies; homemade Mab-6D10 against PRRSV-N protein, or anti-β-tubulin Mab (Transgene). Specific binding between antibodies and targets was detected using HRP-conjugated goat anti-rabbit IgG (Jackson ImmuneResearch) or HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) and visualized using enhanced chemiluminescent (ECL) substrate-based detection (Bio-Rad Laboratories, Hercules, CA, USA). Chemiluminescence signal acquisition was conducted using a ChemiDoc MP Imaging System (Bio-Rad Laboratories) with analysis conducted using the Image Lab software (version 5.1, Bio-Rad Laboratories).

Whole cell lysates of CRL-2843 cells with or without pIFN-α treatment were prepared by addition to cells of 1× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA). Lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [25 (link)] then separated proteins were transferred to PVDF membranes as previously reported [26 (link)]. Membranes were probed with homemade anti-porcine ISG15 (pISG15) Mab-3D5E6 or anti-β-tubulin Mab (Transgene, Beijing, China). Specific binding interactions between antibodies and corresponding targets were detected using HRP-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA) and visualized using enhanced chemiluminescent (ECL) substrate-based detection (Bio-Rad Laboratories). Chemiluminescence signal acquisition was conducted using a ChemiDoc MP Imaging System (Bio-Rad Laboratories) and data were analyzed using ImageLab software (Version 5.1, Bio-Rad Laboratories).

Whole cell lysates of BM-DCs, Vero cells, HEK-293T cells, and cells of the HEK-293T-SLA-DR stable cell line were harvested using 1× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA). Next, lysate proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (24 (link)). Separated proteins were then transferred to PVDF membranes as previously described (25 (link)). Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRα (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRβ, anti-β-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA). Specific binding between antibodies and corresponding targets was detected based on binding to HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific). Results were visualized using ECL substrate (Bio-Rad Laboratories), with chemiluminescent signal acquisition conducted using a ChemiDoc MP Imaging System (Bio-Rad Laboratories) and analysis of data conducted using Image Lab software (Version 5.1, Bio-Rad Laboratories).