nf κb dna binding activity  (Thermo Fisher)

Journal: American journal of human genetics

Article Title: Functional characterization of the 9q34.13 locus identifies RAPGEF1 as a candidate gene modulating risk for melanoma and nevi via RAS activation

doi: 10.1016/j.ajhg.2026.08.014

Figure Lengend Snippet: C283T immortalized melanocytes with doxycycline-inducible expression of RAPGEF1 were grown in supplement-free medium for 24hrs and subsequently treated with human EGF (hEGF) protein for the indicated time. A . Active GTP-bound RAP1 was pulled down by RalGDS-RBD beads from whole-cell extract. Pull-down and whole-cell (total RAP) fractions were compared by Western blotting with an antibody against RAP1. B . Active GTP-bound RAS was pulled down by Raf-RBD beads, with the pull-down and whole-cell (total RAS) fractions compared by Western blotting with an antibody against RAS. Active and total AKT and ERK were compared via Western blotting. The intensity of the phospho- versus total-AKT and ERK was determined by the ratio of the band intensity. Figures are representative experiments from three biological replicates.

Article Snippet: RAP1 activation was measured using the RAP1 activation assay kit from Abcam (ab212011) and RAS activation was measured using GST-fusion Raf-1 RBD beads (Sigma, #14863) or RAF-RBD Protein GST Beads (RF02-A, Cytoskeleton).

Techniques: Activation Assay, Expressing, Pull Down Assay, Western Blot

Journal: Cells

Article Title: Repurposing Rilmenidine as a Potential Antimetastatic Therapy Targeting Nischarin in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cells15111032

Figure Lengend Snippet: NISCH agonist rilmenidine reduces cell migration and invasion in vitro. ( a ) Quantification of the number of PDAC cells spread 6 and 24 h after seeding on tissue culture plastic, collagen I or fibronectin-coated surface in the presence of rilmenidine. Results were expressed as the percentage of spread cells out of the total counted per field. ( b ) Quantification of the PDAC cell migration in the wound healing assay after 24 h of treatment with rilmenidine; ( c ) Quantification of the PDAC cell migration in the transwell assay after 48 h of treatment with rilmenidine; ( d ) Level of active GTP-bound Rac1 in PANC-1 cells from the wound healing assay 24 h post wounding in control or 100 μM rilmenidine-treated PANC1 cells, pulled–down from 600 μg lysate treated with 10 μL PAK-PBD beads; 20 ng of His-tagged Rac1 protein control, positive cellular protein control GTPγS loaded lysate, negative cellular protein control GTP loaded lysate; ( e ) Quantification of PDAC cell invasion through collagen I, fibronectin or matrigel in the transwell assay after 72 h of treatment with rilmenidine; ( f ) Representative images of transwell invasion through matrigel coat (right). Scale bar 200 μm. All data are shown as mean ± SD. n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-way ANOVA (Sidak’s multiple comparisons test).

Article Snippet: We pulled-down GTP-bound Rac1 in PANC-1 cells with PAK-PBD affinity beads (Rac1 Activation Assay, Cytoskeleton) 24 h after introduction of multiple wounds in a confluent layer of cells in absence or presence of 100 μM rilmenidine.

Techniques: Migration, In Vitro, Wound Healing Assay, Transwell Assay, Control

Journal: bioRxiv

Article Title: Sphingosine-1-phosphate cross-talks to Notch via a S1PR1-Dll4-MPDZ complex to regulate endothelial barrier function

doi: 10.64898/2026.05.20.726610

Figure Lengend Snippet: a , Immunoblots showing cleaved Notch1 (c-Notch1) and total Notch1 in MVECs treated with S1P for 1-hour ± γ-secretase inhibitor DAPT; quantification below (mean ± SEM). b , Fluorescent micrographs of YFP-Notch1 reporter intensity after S1P treatment; quantification below. Scale bar, 50 μm. c-d , Fold change in HES1 and HEY1 gene expression levels in (c) MVECs after S1P ± DAPT treatment, and in (d) mouse tissues after intravenous injection of S1P ± DAPT. e , Evans blue dye leakage in mouse dermis 1-hour post-injection with DMSO, S1P, or S1P + DAPT. Scale bar, 5 mm. f-i , Quantified dye leakage in (f) dermis, (g) lung, (h) liver, and (i) kidney, measured via absorbance. j , Heat maps of dextran dye diffusion in engineered microvessels ± S1P. Scale bar, 50 μm. k-l , Diffusive permeability in microvessels lined with (k) DAPT-vs. DMSO-treated or (l) NOTCH1 KO vs. SCR KO MVECs (mean ± SD). m , Micrographs of VE-cadherin (magenta), actin (green), and nuclei (blue) staining in microvessels. Scale bar, 50 μm. n-o , Mean corrected intensities of junctional (n) VE-cadherin and (o) actin from (m). p , Rac1 activity (PBD pull-down). Active/total Rac1 quantification below. q , Schematic of canonical Notch signaling. r , HES1 / HEY1 gene expression levels in dominant negative-MAML-GFP (DN-MAML-GFP) normalized to GFP expressing cells. s , Permeability in DN-MAML-GFP vs GFP-lined microvessels ± S1P. t , Schematic of Notch cortical pathway mediated by its transmembrane domain (TMD). u , Permeability in TMD-RFP vs RFP-lined microvessels ± S1P. v , Permeability in TMD-RFP vs RFP-lined microvessels ± clinical S1P receptor degrader fingolimod.

Article Snippet: PAK-PBD beads to bind active Rac1 (PAK-02) were purchased from Cytoskeleton and reconstituted according to the manufacturer’s instructions.

Techniques: Western Blot, Gene Expression, Injection, Diffusion-based Assay, Permeability, Staining, Activity Assay, Dominant Negative Mutation, Expressing

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , b , Addition of FBP to whole cell lysate causes a conformational change in Rac1. a , Whole cell lysates of U-2 OS cells were subjected to LiP–MS. Peptide intensity (amino acids (aa) 6–16) of Rac1 in response to increasing FBP concentrations. b , Responding peptide (red, aa 6–16) highlighted in the GTP-bound structure of Rac1 (PDB ID: 1MH1 ). c , Increased Rac1 activity upon loss of aldolase. Left: representative immunoblot of pull-down assay using GST-PAK-PBD beads to detect active Rac1 in lysates from siRNA treated U-2 OS cells. α-Tubulin was used as a loading control. Right: a quantification of Rac1-GTP over total Rac1. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 5 independent experiments; two-sided one-sample t -test ( P value = 0.0326). d , Efficient Rac1 depletion. Representative immunoblot of U-2 OS cells treated with the indicated siRNAs. β-actin and clathrin heavy chain (CHC) were used as loading controls; N = 3 independent experiments. e , Codepletion of Rac1 does not significantly lower FBP levels in aldolase-knockdown cells. Relative FBP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 3 independent experiments; two-sided one-sample t -test ( P values: (siRAC1) = 0.1494; (siALDOA) = 0.027; (siRAC1 + siALDOA) = 0.105). f – h , Codepletion of Rac1 restores FA numbers and cell size in aldolase knockdown cells. f , Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. g , h , A quantification of FAs per cell ( f ) and cell area ( g ) shown in f . For f – h , data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for g ): (siCtrl versus siRAC1) = 0.0481; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.9979; P values (for h ): (siCtrl versus siRAC1) = 0.0003; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.2799). i , j , Expression of dominant-negative Rac1 in aldolase-depleted cells rescues increased FA numbers ( i ) and cell size ( j ). A quantification of FAs per cell and cell area of U-2 OS cells transfected with control (−) or ALDOA-specific (+) siRNAs in combination with either eGFP or myc-Rac1-T17N. Corresponding images are shown in Extended Data Fig. . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for i ): (siCtrl + GFP versus siCtrl + T17N) = 0.1497; (siCtrl + GFP versus siALDOA + GFP) <0.0001; (siCtrl + GFP versus siALDOA + T17N) = 0.0633; P values (for j ): (siCtrl + GFP versus siCtrl + T17N) = 0.4683; (siCtrl + GFP versus siALDOA + GFP) <0.0001; (siCtrl + GFP versus siALDOA + T17N) = 0.0981). k , l , Expression of constitutively active Rac1 in PFK-depleted cells rescues reduced FA numbers and cell size. Quantifications of FAs per cell ( k ) and cell area ( l ) of U-2 OS cells transfected with control (−) or PFK-specific (+) siRNAs in combination with either eGFP or myc-Rac1-Q61L. Corresponding images are shown in Extended Data Fig. . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for k ): (siCtrl + GFP versus siCtrl + Rac1-Q61L) = 0.0342; (siCtrl + GFP versus siPFK + GFP) = 0.0063; (siCtrl + GFP versus siPFK + Rac1-Q61L) = 0.8904; P values (for l ): (siCtrl + GFP versus siCtrl + Rac1-Q61L) <0.0001; (siCtrl + GFP versus siPFK + GFP) = 0.0116; (siCtrl + GFP versus siPFK + Rac1-Q61L) = 0.0004). n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 25 µm.

Article Snippet: Cleared lysates were collected and incubated with 25 μg of GST-PAK-PBD beads (Cytoskeleton) for 1 h at 4 °C under constant rotation.

Techniques: Activity Assay, Western Blot, Pull Down Assay, Control, Knockdown, Expressing, Dominant Negative Mutation, Transfection

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , PFK depletion decreases the level of active Rac1. Left: Representative immunoblot of pulldown assay using GST-PAK-PBD beads to detect active Rac1 in lysates from U-2 OS cells treated with indicated siRNAs. β-actin was used as loading control. Right: Quantification of GTP-Rac1 over β-actin. Data were normalized to siCtrl and represent mean ± SEM; n = 5 independent biochemical experiments; two-sided One sample t-test ( P value = 0.0043). b-e , Co-depletion of Rac1 restores FA numbers and cell size upon aldolase knockdown also based on alternative Rac1-targeting siRNAs in RPE-1 cells. b , Representative confocal images of paxillin-labeled FAs in RPE-1 cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. c, d , Quantification of FAs per cell and cell area shown in b . Data represent mean ± SEM; n = 3 (c: siRAC1#6, siRAC1#6 + siALDOA), 4 (c, all others), 5 (d: siRAC1#6, siRAC1#6 + siALDOA) or 6 (d, all others) independent experiments; statistical testing by mixed-effects model (REML) with Dunnett’s multiple comparison test ( P values ( c ): (siCtrl vs. siRAC1#5) = 0.6235; (siCtrl vs. siRAC1#6) = 0.4587; (siCtrl vs. siALDOA) = 0.0332; (siCtrl vs. siRAC1#5 + siALDOA) = 0.3327; (siCtrl vs. siRAC1#6 + siALDOA) = 0.9142; P values ( d ): (siCtrl vs. siRAC1#5) = 0.3583; (siCtrl vs. siRAC1#6) = 0.1119; (siCtrl vs. siALDOA) = 0.0006; (siCtrl vs. siRAC1#5 + siALDOA) = 0.8081; (siCtrl vs. siRAC1#6 + siALDOA) = 0.2114). Ns, not significant. e , Efficient Rac1 and aldolase depletion in RPE-1 cells. Immunoblot of RPE-1 cells treated with indicated siRNAs. CHC was used as loading control. N = 1 independent experiment. f , Expression of dominant-negative Rac1 in aldolase-depleted cells rescues FA number and cell size. Representative confocal images of paxillin-labeled FAs in U-2 OS cells transfected with control or ALDOA-specific siRNAs in combination with either eGFP or myc-Rac1-T17N. FA segmentation and cell outlines (red) are shown below. Corresponding quantification is shown in Fig. . g , Expression of constitutively active Rac1 in PFK depleted cells elevates FA number and cell size to normal levels. Representative confocal images of paxillin-labeled FAs in U-2 OS cells transfected with control or PFK-specific siRNAs in combination with either eGFP or myc-Rac1-Q61L. FA segmentation and cell outlines (red) are shown below. Corresponding quantification is shown in Fig. . Ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 25 µm.

Article Snippet: Cleared lysates were collected and incubated with 25 μg of GST-PAK-PBD beads (Cytoskeleton) for 1 h at 4 °C under constant rotation.

Techniques: Western Blot, Control, Knockdown, Labeling, Comparison, Expressing, Dominant Negative Mutation, Transfection

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , b , Codepletion of Rac1 in aldolase-treated cells normalizes F-actin organization and cell protrusion. a , Representative confocal images of the phalloidin-labelled F-actin cytoskeleton in U-2 OS cells treated with indicated siRNAs. Zoom-ins of orange boxes are shown. b , A quantification of the protrusive area of U-2 OS cells treated with indicated siRNAs. Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl versus siRAC1) = 0.5925; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.0825). Corresponding images are shown in Extended Data Fig. . c , d , Codepletion of Rac1 in aldolase-depleted cells restores cell spreading to normal levels. c , Representative confocal images of phalloidin-labelled U-2 OS cells treated with indicated siRNAs. The cells were seeded and fixed after indicated time points. d , A quantification of cell spreading shown in c . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl versus siRAC1) = 0.7944; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.6024). e , f , Glucose triggers cell spreading. e , Representative images of phalloidin-labelled RPE-1 cells incubated with the indicated glucose concentrations. The cells were seeded and fixed after 2 h. f , A quantification of cell spreading shown in e . Data represent mean ± s.e.m.; n = 3 independent experiments; two-sided unpaired t -test ( P value = 0.0012). g , h , Increased Rac1 activity upon glucose. g , Representative immunoblot of a pull-down assay using GST-PAK-PBD beads to detect active GTP-Rac1 in lysates from untreated or glucose exposed RPE-1 cells. β-actin was used as a loading control. h , A quantification of GTP-Rac1 over total Rac1. The data were normalized to 0 mM glucose condition and represent mean ± s.e.m.; n = 3 independent experiments; two-sided one-sample t -test ( P value = 0.0228). n.s., not significant; * P < 0.05, ** P < 0.01, **** P < 0.0001. Scale bars, 25 µm for a and c ; 10 µm for e .

Article Snippet: Cleared lysates were collected and incubated with 25 μg of GST-PAK-PBD beads (Cytoskeleton) for 1 h at 4 °C under constant rotation.

Techniques: Incubation, Activity Assay, Western Blot, Pull Down Assay, Control