ap conjugated goat anti mouse ig  (SouthernBiotech)

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: KEAP1 is the direct target of TUS. (A) Scheme of TUSP synthesis. Reagents and reaction conditions, a, HCl, H 2 O, THF, rt, 12 h. b, 2,4,6-trichloro-benzoyl chloride, triethylamine, DMAP, DCM, rt, 12 h. (B-C) The inhibition of TUS or TUSP on TNF-α and NO induced by LPS. RAW 264.7 macrophages were treated with 1 μg/mL LPS and 6.3, 12.5, 25, 50 μM TUS or TUSP for 24 h, the level of TNF-α and NO were detected and compared. (D) KEAP1 was identified as the direct binding target of TUS. The cell lysate of Beas-2B cells was incubated with TUSP or biotin (negative control) for 1 h, and pulled down with streptavidin-conjugated beads. Subsequently, the bound proteins were detected using SDS-PAGE gel, followed by silver staining for visualization and LC-MS/MS analysis for identification. (E-F) The binding of TUS with KEAP1 in Beas-2B cells and recombinant human KEAP1 protein were detected using Western blot. Cell lysate or recombinant human KEAP1 were incubated with TUSP or biotin (negative control) for 1 h, and pulled down with streptavidin-conjugated beads, KEAP1 levels were detected with Western blot. (G) CETSA suggested that TUS improved the thermal stability of KEAP1 protein at different temperatures. Beas-2B cells were treated with 20 μM TUS or DMSO (negative control) for 8 h, then the thermal stability of KEAP1 was detected with CETSA assay. (H) DARTS suggested that TUS improved the enzymatic stability of KEAP1 against pronase E. Cell lysate was incubated with 0, 5, 10, 20, 40 μM TUS or DMSO (negative control) for 1 h, followed by addition of 500 ng/mL pronase E. KEAP1 levels were detected with Western blot. (I) SPR assay showed the kinetics of increasing concentrations of TUS binding to KEAP1.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: Inhibition, Binding Assay, Incubation, Negative Control, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Recombinant, Western Blot, SPR Assay

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: Cys434 is the crucial amino acid for the binding of TUS to KEAP1. (A) IAA blocked the binding of TUS to KEAP1. Cell lysate was incubated with 200 μM IAA or TUS for 1 h, followed by 20 μM TUSP or biotin (negative control) for another 1 h. Target proteins were pulled down with streptavidin-conjugated beads, and KEAP1 levels were detected with Western blot. (B) LC-MS/MS indicated that Cys434 was the binding site of TUS to KEAP1. The human recombinant protein KEAP1 was incubated with TUS for 1 h, followed by digestion and mass spectrometry analysis to identify the binding sites of TUS. (C) C434A mutation abolished the binding activity between TUS and KEAP1. (D) DARTS suggested that TUS had no effect on the enzymatic stability of KEAP1 against pronase E. (E) CETSA indicated that TUS had no effect on the thermal stability of the KEAP1 protein after the C434A mutation. (F) The binding of TUS to WT or C434A mutant recombinant human KEAP1 proteins was detected using pulldown assay and Western blot analysis. (G) Classification of highly reactive cysteines and inducers of KEAP1.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: Binding Assay, Incubation, Negative Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Recombinant, Mass Spectrometry, Mutagenesis, Activity Assay