Anti mouse irdye 680 secondary antibodies

American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer Inc. (Boston, MA) were the suppliers for radiolabelled [¹⁴C]-AA (2.8-10 mCi/mmol, radiochemical purity > 98%). The anti-β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). In addition, western blot protocols utilized anti-rabbit IRDye-800 and anti-mouse IRDye-680 secondary antibodies sourced from LI-COR Biosciences (Lincoln, NE). The recombinant human IL-1β was acquired through R&D Systems, Inc. (Minneapolis, MN). Integrated DNA Technologies (San Diego, CA) supplied the individually synthesized custom oligonucleotide primers used in RT-qPCR analysis. All further chemicals, kits, and molecular biological reagents were obtained from reputable scientific manufacturers and stored appropriately to maintain substance integrity and stability.

CHO-K1 cells were transfected with control or the plasmids as indicated in the figure legends. After 24 h, transfected cells in 6-well plates were collected and lysed in 200 µl lysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl₂, 1% Triton X-100, and Calbiochem 1× protease inhibitor cocktail set I). The cell lysates were cleared of debris by centrifugation, and 100 µl of the lysates was mixed with 100 µl 2× SDS loading buffer (60 mM Tris-Cl, pH 6.8, 0.2% SDS, 25% glycerol, 0.01% bromophenol blue). The samples were loaded onto a Bio-Rad 7.5% mini-Protean TGX gel for Western blotting. After electrophoresis, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl) for 2 h at room temperature (RT). The blots were washed with TBS and incubated with primary antibodies (anti-gB or anti-gH/gL) overnight at 4°C. Anti-rabbit IRDye800 or anti-mouse IRDye680 secondary antibodies (Li-Cor Bioscience, Lincoln, NE) were added to the membranes at a dilution ratio of 1:10,000, and incubation was continued for 1 h at RT. Protein bands on the membrane were visualized with the Odyssey Fc Western blotting imager using Image Studio version 2.0 (Li-Cor Bioscience, Lincoln, NE).