Beta actin rabbit polyclonal antibody

A rabbit anti-PEDV-N protein polyclonal antibody was generated in the laboratory [24 (link)]. A beta-actin rabbit polyclonal antibody was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Alexa 488-conjugated goat anti-rabbit IgG antibody and 4’,6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific, Inc. (San Hose, CA, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Apex Bio Technology, Inc. (Houston, TX, USA). A soluble TMB Substrate Solution (TMB) was obtained from Tiangen (Beijing, China). HiScript II Reverse Transcriptase and 2×Taq Master Mix were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). An RNA extraction kit was obtained from PuDi Biotech Co., Ltd. (Shanghai, China). LysoSensor™ Yellow/Blue DND-160 was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Magic Red^® Cathepsin L Assay Kit and Magic Red^® Cathepsin B Assay Kit were obtained from Immunochemistry Technologies, LLC. (Davis, CA, USA). BBM, FAN, and +FAN were purchased from Selleck Chemicals (Houston, TX, USA). NH4Cl was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Total protein was extracted from tissues and cells and lysed in RIPA buffer (Beyotime, China) mixed with protease/phosphatase inhibitors (Sigma-Aldrich, USA). Samples were collected via centrifugation at 13,000 g and 4 °C for 5 min. The supernatants were boiled at 100 °C for 5 min in loading buffer. A BCA protein assay (Thermo Scientific, USA) was used to measure protein concentrations. Equal amounts of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with primary antibody (HIF-1α mouse monoclonal antibody, 1:2000, Novus; TNF alpha rabbit polyclonal antibody, 1:1000, Abcam; MCP-1 mouse monoclonal antibody, 1:1000, Novus; beta tubulin rabbit polyclonal antibody, 1:1000, Proteintech; GAPDH rabbit polyclonal antibody, 1:1000, Proteintech; beta actin rabbit polyclonal antibody, 1:1000, Proteintech). An Alexa Fluor 680/790-labeled goat anti-rabbit/goat anti-mouse IgG antibody (1:25,000, LI-COR Biosciences, USA) was used as the secondary antibody. An Odyssey infrared imaging system (LI-COR Biotechnology, USA) was employed to visualize the blots.