af2006  (Affinity Biosciences)

Journal: Biology

Article Title: Kukoamine B Inhibits EMT in Lung Adenocarcinoma Cells by Regulating Intracellular PD-L1-Mediated p65 Nuclear Translocation

doi: 10.3390/biology15050435

Figure Lengend Snippet: Intracellular PD-L1 interaction with p65 and promotion of p65 nuclear translocation. ( A , B ) Co-immunoprecipitation (Co-IP) experiments demonstrating the interaction between PD-L1 and phosphorylated p65 (p-p65) in PD-L1 overexpressing A549 and H1299 cells compared to control cells (see for related original images); ( C , D ) Western blot analysis showing increased nPD-L1 and nuclear phosphorylated p65 (np-p65) levels in PD-L1 overexpressing A549 and H1299 cells (see for related original images); ( E ) confocal immunofluorescence imaging illustrating nPD-L1 and np-p65 localization in A549 and H1299 cells (scale bar: 10 μm). Significance levels are indicated as * p < 0.05; ** p < 0.01. All experiments included three biological replicates ( n = 3). Abbreviations: NC, empty vector control; OE, overexpression vector.

Article Snippet: In this study, the antibodies utilized were as follows: Normal Rabbit IgG (2729S) and p65 (8242S) rabbit monoclonal antibodies from CST; PD-L1/CD274 (28076-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), Vimentin (10366-1-AP), Lamin B1 (12987-1-AP), GAPDH (10494-1-AP), and HRP Mouse Anti-Rabbit IgG Light Chain Specific (SA00001-7L) from Proteintech; Phospho-p65 (AF2006) from Affinity; and secondary antibody HRP Goat Anti-Rabbit IgG from Abclonal (AS014).

Techniques: Translocation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Immunofluorescence, Imaging, Plasmid Preparation, Over Expression

Journal: Biology

Article Title: Kukoamine B Inhibits EMT in Lung Adenocarcinoma Cells by Regulating Intracellular PD-L1-Mediated p65 Nuclear Translocation

doi: 10.3390/biology15050435

Figure Lengend Snippet: Molecular docking and MD simulations elucidate the binding association between PD-L1 and p65. ( A – E ) ZDOCK docking predictions illustrating the interaction between PD-L1 (shown in green) and p65 (in cyan), corresponding to the top 1–5 ranked docking conformations; ( F ) RMSD analysis of the fifth-ranked PD-L1–p65 complex during molecular dynamics simulations—the black line represents the entire PD-L1/p65 complex, the green line represents PD-L1, and the red line represents p65; ( G ) B-factor analysis depicting the flexibility of the protein complex, with deep blue line indicating the most rigid regions and red line showing the most flexible regions; ( H ) structural snapshots of the complex at different time points during the simulation: 40 ns (green), 80 ns (cyan), 120 ns (magenta), and 160 ns (yellow); ( I ) hydrogen bond fluctuations at the PD-L1–p65 interface throughout the MD simulation.

Article Snippet: In this study, the antibodies utilized were as follows: Normal Rabbit IgG (2729S) and p65 (8242S) rabbit monoclonal antibodies from CST; PD-L1/CD274 (28076-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), Vimentin (10366-1-AP), Lamin B1 (12987-1-AP), GAPDH (10494-1-AP), and HRP Mouse Anti-Rabbit IgG Light Chain Specific (SA00001-7L) from Proteintech; Phospho-p65 (AF2006) from Affinity; and secondary antibody HRP Goat Anti-Rabbit IgG from Abclonal (AS014).

Techniques: Binding Assay

Journal: Biology

Article Title: Kukoamine B Inhibits EMT in Lung Adenocarcinoma Cells by Regulating Intracellular PD-L1-Mediated p65 Nuclear Translocation

doi: 10.3390/biology15050435

Figure Lengend Snippet: Binding Free Energy and Interface Analysis Unveils the Strong Binding Affinity of the PD-L1/p65 Complex. ( A , B ) A 3D visualization showing the interaction residues of PD-L1 (green) and p65 (cyan), with hydrogen bonds represented as solid magenta lines—the electrostatic surfaces of residues involved in hydrophobic interactions are also depicted; ( C ) LigPlot+ analysis illustrating the interactions between PD-L1 (chain A) and p65 (chain B), where hydrogen bonds are shown as green dashed lines, labeled with distances, and hydrophobic contacts are indicated by red arcs pointing toward the interacting residues; ( D ) bar chart displaying the contributions of various energetic components to the binding free energy of the PD-L1–p65 complex, as calculated using the MM/GBSA method, including components such as VDWAALS (van der Waals interactions), EEL (electrostatic interactions), EGB (polar solvent interactions), ESURF (non-polar solvation energy), GGAS (gas-phase energy: VDWAALS + EEL), and GSOLV (solvation energy: EGB + ESURF); ( E ) per-residue energy contributions for individual residues in the PD-L1–p65 complex, with the bar chart indicating the stabilization contributions (negative values) of each residue to the binding free energy (kcal/mol); ( F ) heat map illustrating per-residue energy contributions throughout the MD simulation, with residues from PD-L1 (chain A) and p65 (chain B) displayed, and a color scale ranging from blue (stabilizing contributions) to red (destabilizing contributions) providing insights into the interaction strength and stability across simulation frames; ( G ) Gibbs free energy landscape of the PD-L1–p65 complex, represented as a heat map of RMSD versus the radius of gyration (GYRATE), where blue regions correspond to low-energy, stable states and red regions indicate higher energy states; ( H ) Gibbs energy landscape focusing on specific residues within the PD-L1 (19F-132A) and p65 (296H-314K) domains, highlighting regions of significant stability; ( I ) detailed structural view of the binding interface between PD-L1 and Kukoamine B, with the Kukoamine B-binding residues on PD-L1 shown as electrostatic potential surfaces.

Article Snippet: In this study, the antibodies utilized were as follows: Normal Rabbit IgG (2729S) and p65 (8242S) rabbit monoclonal antibodies from CST; PD-L1/CD274 (28076-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), Vimentin (10366-1-AP), Lamin B1 (12987-1-AP), GAPDH (10494-1-AP), and HRP Mouse Anti-Rabbit IgG Light Chain Specific (SA00001-7L) from Proteintech; Phospho-p65 (AF2006) from Affinity; and secondary antibody HRP Goat Anti-Rabbit IgG from Abclonal (AS014).

Techniques: Binding Assay, Labeling, Solvent, Residue

Journal: Biology

Article Title: Kukoamine B Inhibits EMT in Lung Adenocarcinoma Cells by Regulating Intracellular PD-L1-Mediated p65 Nuclear Translocation

doi: 10.3390/biology15050435

Figure Lengend Snippet: Kukoamine B inhibits p65 nuclear translocation by disrupting the PD-L1–p65 interaction. ( A , B ) Co-immunoprecipitation (Co-IP) analysis showing the interaction between PD-L1 and p65 in PD-L1-overexpressing and control A549 and H1299 cells after 24 h treatment with Kukoamine B (see for related original images); ( C , D ) Western blot analysis demonstrating the levels of nPD-L1 and nuclear p65 (np65) in PD-L1-overexpressing A549 and H1299 cells following 24 h of Kukoamine B treatment (see for related original images). Statistical significance is indicated as * p < 0.05; ** p < 0.01; ns indicates no significance ( p > 0.05). All experiments included three biological replicates ( n = 3). Abbreviations: NC, empty vector control; OE, overexpression vector.

Article Snippet: In this study, the antibodies utilized were as follows: Normal Rabbit IgG (2729S) and p65 (8242S) rabbit monoclonal antibodies from CST; PD-L1/CD274 (28076-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), Vimentin (10366-1-AP), Lamin B1 (12987-1-AP), GAPDH (10494-1-AP), and HRP Mouse Anti-Rabbit IgG Light Chain Specific (SA00001-7L) from Proteintech; Phospho-p65 (AF2006) from Affinity; and secondary antibody HRP Goat Anti-Rabbit IgG from Abclonal (AS014).

Techniques: Translocation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Plasmid Preparation, Over Expression

Journal: NPJ Biofilms and Microbiomes

Article Title: Role of intestinal SCFAs homeostasis in the hepatoprotective effect of Clostridium butyricum in T2DM

doi: 10.1038/s41522-025-00824-5

Figure Lengend Snippet: A The relative phosphorylation levels of NF-κB in the liver of mice by IF staining. Scale bars were 50 µm (above) and 20 µm (below). B The relative phosphorylation levels of NF-κB-p65 in the liver of NaB treated mice by Western Blot. C The relative phosphorylation levels of NF-κB-p65 in NaB treated HepG2 cells by IF staining. Scale bars were 20 µm. D The relative phosphorylation levels of NF-κB-p65 in NaB treated HepG2 cells by Western Blot. E Effects of TGR5 knockdown on the relative phosphorylation levels of NF-κB-p65 in NaB treated HepG2 cells by IF staining. Scale bars were 20 µm. F Effects of TGR5 knockdown on the relative phosphorylation levels of NF-κB-p65 in NaB treated HepG2 cells by Western Blot. G Luciferse activity of NF-κB promoter in HepG2 cells. H EMSA of TGR5 blinding to the NF-κB promoter in HepG2 cells. Each bar represents the mean ± SEM for mice groups of six and cell groups of three. ** P < 0.01, compared to db/m or NG; ## P < 0.01, compared to db/db or HG; $ P < 0.05, $$ P < 0.01, compared to HG + FFA; % P < 0.05, %% P < 0.01, compared to HG + FFA + NaB + TGR5-shRNA-Veh.

Article Snippet: Antibodies against p-NF-κB-p65 (AF2006) and p-IκB-α (AF2002) were sourced from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Phospho-proteomics, Staining, Western Blot, Knockdown, Activity Assay, shRNA

Journal: NPJ Biofilms and Microbiomes

Article Title: Role of intestinal SCFAs homeostasis in the hepatoprotective effect of Clostridium butyricum in T2DM

doi: 10.1038/s41522-025-00824-5

Figure Lengend Snippet: A Effects of PMA and PDTC on the levels of liver function indicators in NaB treated HepG2 cells. B The levels of LDH in PMA and PDTC treated HepG2 cells. C The relative phosphorylation levels of NF-κB-p65 in HepG2 cells by IF staining. Scale bars were 20 µm. D The relative phosphorylation levels of NF-κB-p65 in HepG2 cells by Western Blot. E The relative levels of inflammatory factors in DMEM medium of HepG2 cells. F Effects of PMA and PDTC on the relative levels of inflammatory factors in HepG2 cells by Western Blot. G The protein levels of Collagen Ⅳ and Laminin in PMA and PDTC treated HepG2 cells by IF staining. Scale bars were 20 µm. Each bar represents the mean ± SEM for cell groups of three. ** P < 0.01, compared to HG + FFA; & P < 0.05, && P < 0.01 compared to HG + FFA+NaB-H.

Article Snippet: Antibodies against p-NF-κB-p65 (AF2006) and p-IκB-α (AF2002) were sourced from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Phospho-proteomics, Staining, Western Blot

Journal: NPJ Biofilms and Microbiomes

Article Title: Role of intestinal SCFAs homeostasis in the hepatoprotective effect of Clostridium butyricum in T2DM

doi: 10.1038/s41522-025-00824-5

Figure Lengend Snippet: Schematic diagram depicting the possible mechanism of CB against hepatitis in T2DM through activating hepatic TGR5 via intestinal butyric acid, leading to the inactivated IκB-α/β-arrestin2/NF-κB signaling pathway.

Article Snippet: Antibodies against p-NF-κB-p65 (AF2006) and p-IκB-α (AF2002) were sourced from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: