anti β tubulin mouse monoclonal antibody  (Proteintech)

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: Metabolic reprogramming of cumulus–oocyte complexes (COCs). (a) Flowchart for metabolomics analysis. (b) PCA of Ctrl and LP group. (c) Identified differential metabolites, blue squares mean down regulated metabolites, yellow triangles mean up regulated metabolites. (d) Heatmap of differential metabolites. (e) KEGG pathway enrichment analysis of COCs from Ctrl and LP group. MT: mitochondria; SCSFAs: short chain saturated fatty acids; LCSFAs: long chain saturated fatty acids. (f) The most enriched metabolites in significantly differential KEGG pathways, metabolites are represented in KEGG gene numbers. (g) NAD + and NADH abundance of COCs in LP (n = 3) group relative to Ctrl (n = 3) group. (h) The level of NAD + and NADH in ovaries from Ctrl (n = 4) and LP (n = 3) group. (i) Ovaries were separated from Ctrl and LP group for western blotting (WB), β-Tubulin was used as control. Statistically significant differences between the Ctrl and LP groups were determined using Tukey's test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The antibodies employed were as follows: rabbit anti-BMAL1 (Cell signalling, 14020; 1:1000 dilution), mouse anti-Beta Tubulin (Proteintech, 66240-1-Ig; 1:1000 dilution), rabbit anti-NAMPT (Proteintech, 11776-1-AP; 1:1000 dilution), rabbit anti-Cytokeratin 18 (Proteintech, 10830-1-AP; 1:1000 dilution), rabbit anti-FHSR (Proteintech, 22665-1-AP; 1:1000 dilution), rabbit anti-VIMENTIN (Proteintech, 10366-1-AP; 1:1.000 dilution), rabbit anti-VDAC1/Porin (Proteintech, 55259-1-AP; 1:1000 dilution), rabbit anti-SIRT3 (Proteintech, 10099-1-AP; 1:1000 dilution), rabbit anti-SOD2/MnSOD (Abcam, ab68155; 1:1000 dilution), rabbit anti-SOD2/MnSOD (acetyl K68) (Abcam, ab137037; 1:1000 dilution).

Techniques: Western Blot, Control

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: Disruption of NAMPT oscillated expression through NAMPT after long photoperiod exposure. (a) Ovaries were obtained at different ZT from Ctrl and LP group for WB, β-Tubulin was used as control. (b) The relative expression of BMAL1 at different ZT based on ZT0 to show the dynamic changes of Ctrl and LP group (n = 3). (c) The relative expression of NAMPT at different ZT based on ZT0. (d) WB of ovaries from Ctrl and LP group at each ZT (n = 3). (e) BMAL1 expression of LP group relative to Ctrl at each ZT (n = 3). (f) NAMPT expression of LP group relative to Ctrl at each ZT (n = 3). (g) Linear correlation of NAMPT and BMAL1 expression in Ctrl and LP group, which were showed for calculated expression based on its fold change to raw expression of ZT0. (h) Genomic views of BMAL1 CUT&Tag-seq assay enrichment at the promoters of the Nampt . (i) The non-canonical E-box motifs in the promoter and first intron of the Nampt gene. Chromatin immunoprecipitation (ChIP) qPCR was used to show that BMAL1 physically and specifically associate to the E-boxes on the Nampt promoter, since the comparative analysis with 1% input as a reference revealed a significant decrease in fold change at the LP group site. (j) BMAL1-NAMPT-SIRT3-Deacetylation SOD2 axis. Statistically significant differences between the Ctrl and LP groups were determined using Tukey's test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The antibodies employed were as follows: rabbit anti-BMAL1 (Cell signalling, 14020; 1:1000 dilution), mouse anti-Beta Tubulin (Proteintech, 66240-1-Ig; 1:1000 dilution), rabbit anti-NAMPT (Proteintech, 11776-1-AP; 1:1000 dilution), rabbit anti-Cytokeratin 18 (Proteintech, 10830-1-AP; 1:1000 dilution), rabbit anti-FHSR (Proteintech, 22665-1-AP; 1:1000 dilution), rabbit anti-VIMENTIN (Proteintech, 10366-1-AP; 1:1.000 dilution), rabbit anti-VDAC1/Porin (Proteintech, 55259-1-AP; 1:1000 dilution), rabbit anti-SIRT3 (Proteintech, 10099-1-AP; 1:1000 dilution), rabbit anti-SOD2/MnSOD (Abcam, ab68155; 1:1000 dilution), rabbit anti-SOD2/MnSOD (acetyl K68) (Abcam, ab137037; 1:1000 dilution).

Techniques: Disruption, Expressing, Control, Chromatin Immunoprecipitation, ChIP-qPCR

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: NMN supplementation alleviates long photoperiod induced mitochondria dysfunction through SOD2. (a) Representative flow cytograms of cellular mitochondrial membrane potential by using JC-1 staining, from primary granulosa cells (GCs) of Ctrl + NMN, Ctrl, LP and LP + NMN groups. (b) Quantitative analysis of the ratio of red to green fluorescence in primary GCs (n = 4); red fluorescence represents JC-1 aggregates, and green fluorescence represents monomeric JC-1. (c) Representative flow cytometry histograms showing ROS detection using DCFH-DA staining in GCs from four groups. (d) Quantitative analysis of ROS in GCs from four groups (n = 4). (e) The level of SOD in ovaries from four groups (n = 8). (f) The level of MDA in ovaries from four groups (n = 8). (g) The OCR of four groups was measured with the Mito Stress test under basal conditions and after administering oligomycin, FCCP, and rotenone/antimycin A (Rot/AA) to modulate mitochondrial respiratory chain proteins. Basal respiration, maximal respiration and ATP production of Ctrl + NMN, Ctrl, LP and LP + NMN group (n = 10) were calculated with normalised data obtained. (h) Ovaries were obtained for WB, β-Tubulin was used as control. The expression levels of SOD2, Ace-SOD2 are shown (n = 9). Statistically significant differences between the Ctrl + NMN, Ctrl, LP and LP + NMN groups were determined using one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The antibodies employed were as follows: rabbit anti-BMAL1 (Cell signalling, 14020; 1:1000 dilution), mouse anti-Beta Tubulin (Proteintech, 66240-1-Ig; 1:1000 dilution), rabbit anti-NAMPT (Proteintech, 11776-1-AP; 1:1000 dilution), rabbit anti-Cytokeratin 18 (Proteintech, 10830-1-AP; 1:1000 dilution), rabbit anti-FHSR (Proteintech, 22665-1-AP; 1:1000 dilution), rabbit anti-VIMENTIN (Proteintech, 10366-1-AP; 1:1.000 dilution), rabbit anti-VDAC1/Porin (Proteintech, 55259-1-AP; 1:1000 dilution), rabbit anti-SIRT3 (Proteintech, 10099-1-AP; 1:1000 dilution), rabbit anti-SOD2/MnSOD (Abcam, ab68155; 1:1000 dilution), rabbit anti-SOD2/MnSOD (acetyl K68) (Abcam, ab137037; 1:1000 dilution).

Techniques: Membrane, Staining, Fluorescence, Flow Cytometry, Control, Expressing

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