Horseradish peroxidase labeled goat anti rabbit igg h l

Selenium-enriched soybeans were purchased from Enshi Se-Run Health Tech Development Co., Ltd. (Enshi city, China), while L-SeMet was obtained from Shanghai Macklin Biochemical Co., Ltd. Na₂SeO₃ was purchased from Sigma Chemical Co. (St. Louis, MO, United States). Xi’an Ruidi Biotechnology Co., Ltd., supplied the honeysuckle extract. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), GSH, and GSH-Px kits were obtained from Nanjing Jiancheng Bioengineering Institute. The IL-1β, IL-6, TNF-α, GSH, and GSH-Px ELISA kits were acquired from Jiangsu Meibiao Biotechnology Co., Ltd., while the BCA protein concentration assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. The skim milk powder was supplied by Becton, Dickinson, and Company. Furthermore, the PVDF membrane, TBST, NF-κB p65 rabbit monoclonal antibody, phospho-IκBα (Ser32/36) rabbit polyclonal antibody, α-Tubulin rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L), Western secondary antibody dilution, and BeyoECL Star (extra super-sensitive enhanced chemiluminescence kit) were purchased from Beyotime Biotechnology.

The membrane proteins were characterized by polyacrylamide gel electrophoresis (SDS-PAGE). The membrane proteins of the MM vesicles and MM/RAPNPs were extracted by Cell Total Protein Extraction kits. The extracted membrane proteins were run on a 4-12% Bis-Tris 10-well minigel in running buffer using a Bio-Rad electrophoresis system at 75 V for 0.5 h and then at 140 V for 1 h. Finally, the resulting polyacrylamide gel was stained with SimplyBlue overnight for visualization.
Furthermore, the integrin α4β1 and CD47 contents in RAW264.7 cells, MMs, MM/RAPNPs and RAPNPs were determined by western blot analysis. The total protein of the lysis solution from 1 × 10⁷ RAW264.7 cells, RAPNPs, MMs extracted from 1 × 10⁷ cells and the subsequent MM/RAPNPs were extracted by Cell Total Protein Extraction kits and used for measurements. Samples underwent electrophoresis on a 10% SDS-polyacrylamide gel and were transferred to a polyvinylidene difluoride membrane (Millipore, USA). Then, the membranes were treated with primary antibodies against α4 (anti-integrin α4, 8440S, CST), β1 (anti-integrin β1, 34971, CST), and CD47 (anti-CD47 antibody, ab175388, Abcam), followed by horseradish peroxidase-labeled goat/anti-rabbit IgG (H+L) (Beyotime, Jiangsu, China). The protein signals were measured by the enhanced chemiluminescence method using a ChemiDoc MP imaging system (Bio-Rad, USA).

Protein lysates from the cell lines were prepared in lysis buffer and centrifuged at 4 °C and 12 000 g. Western blotting was performed according to a previously described procedure (Ye et al., 2015) using rabbit anti‐human IgG1 (ABIN2862696), anti‐human CD47 (ab108415; Abcam, Cambridge, England), anti‐coxsackie adenovirus receptor (CAR; ab100811; Abcam, Cambridge, England), and anti‐CD46 antibody (ab108307; Abcam, Cambridge, England) as the primary antibodies. The secondary antibody was horseradish peroxidase‐labeled goat anti‐rabbit IgG (H+L) (Beyotime, Shanghai, China). The expression of each band was quantitatively analyzed using the image lab™ software (Bio‐Rad, CA, USA).