Anti biotin antibody

Lipopolysaccharide (LPS; purity ≥98%) and dexamethasone sodium phosphate (DXM; purity ≥99%) were obtained from Solarbio Science & Technology (Beijing, China). Poly (I:C) (purity ≥99%) was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). High-quality specific agonists or antagonists of STING, TBK1, GSK3β, and A_2AR were purchased from Topscience Co., Ltd. (Shanghai, China). Primary anti-A_2AR and anti-hemagglutinin (HA) antibodies were obtained from Proteintech Co., Ltd. (Wuhan, China). Anti-biotin antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-phospho-GSK3β (p-GSK3β), anti-GSK3β, anti-phospho-STING (p-STING), anti-STING, anti-phospho-TBK1 (p-TBK1), anti-TBK1, anti-phospho-IRF3 (p-IRF3), anti-IRF3, anti-phospho-NF-κB (p-NF-κB), anti-NF-κB, anti-IL-6, anti-β-actin, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Alexa Fluor secondary antibodies were purchased from Servicebio (Wuhan, China).

For the biomolecular fluorescence complementation assay (BiFC) assay, HEK-293 cells were cotransfected with different BiFC constructs as previously described (Xu et al. 2015 (link), Khatiwada et al. 2023 (link)). Briefly, HEK-293 cells transfected with different BiFC constructs in the absence or presence of TA1 or AT1 for 48 h the cells and green fluorescence was observed under an Olympus microscope. Immunofluorescence was used to study the subcellular localization of the peptides in LNCaP and CWR-22Rv1 cells, which were treated with biotin-TA1 or biotin-AT1. Anti-biotin antibody (Santa Cruz Biotechnology) was used in LNCaP cells and CWR-22Rv1 cells to detect the peptides and anti-AR (Cell Signaling Technology), anti-AR-V7 (Cell Signaling Technology), and anti-TM4SF3 (Fisher Scientific) antibodies were also used in CWR-22Rv1 cells to measure colocalization. Secondary antibodies used were anti-rabbit secondary conjugated to Alexa Fluor 488 and anti-mouse secondary antibody conjugated to Alexa Fluor 546 (both from Life Technologies). Cells were also stained with DAPI for the detection of nuclei and observed on a Leica confocal microscope.