Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 1 Higher ERK1/2 and p38 phosphorylation levels in dormant compared with aggressive T-ALL tumor samples inversely correlate with MKP-1 expression levels. (a) Detection of total and phosphorylated levels of ERK1/2, p38 and MKP-1 by western blot analysis of lysates from dormant or aggressive tumor samples formed by s.c. injection of MOLT-3 cells in NOD/SCID mice in the absence or the presence of exogenous bFGF (100 ng/ml), respectively.21 Membranes were probed with anti-a-tubulin as a loading control. Five representative samples per group are shown. (b) Columns report the mean values±s.d. of phosphorylated ERK (P-ERK)/ERK, P-p38/p38 (left), MKP-1/a-tubulin (right) ratios in all samples analyzed (n ¼ 10 per group). Samples were normalized to the ratio measured in the weakest one of the series, which was set at 1. Signal intensity was measured using a Bio-Rad XRS chemioluminescence detection system. *Po0.05 compared with dormant tumor values. (c) Expression of MKP-1 mRNA is similar in growing compared with dormant tumors. Total RNA was extracted from growing and dormant tumors (n ¼ 7 samples per group) and MKP-1 expression was determined by quantitative PCR. Columns represent mean values±s.d. MOLT-3 cells in vitro were used as reference sample to calculate the relative expression indicated in the panels. (d) MKP-1 is broadly expressed in T-ALL cell lines. Western blot analysis of MKP-1 in a panel of T-ALL cell lines; a-tubulin was used as loading control.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Phospho-proteomics, Expressing, Western Blot, Injection, Control, Real-time Polymerase Chain Reaction, In Vitro

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 2 Notch3 signalling regulates MKP-1 expression. (a) Treatment of T-ALL cell lines with GSIs lowers MKP-1 protein expression. MOLT-3, Jurkat, DND41 and CEM cells were treated for 72 h with CompE (10 mM) or solvent (dimethylsulphoxide (DMSO)) before western blot analysis. Columns represent mean values±s.d. of n ¼ 3 experiments. Cells cultivated in vitro in the presence of DMSO were used as reference sample to calculate the relative expression indicated in the panels. (b) Forced Notch3 ICD expression affects MKP-1 protein expression in MOLT-3 cells. Western blot analysis of MOLT-3 cells transfected by a Notch3 ICD expression plasmid (N3-ICD) versus control cells (enhanced green fluorescent protein (EGFP)). Density values were normalized to the ratio measured in the control sample, which was set at 1. (c) Notch3 silencing reduces MKP-1 expression and activates p38 in T-ALL cell lines. Left panel, western blot analysis of MOLT-3 cells transduced by lentiviral vectors encoding two different Notch3-specific shRNA (shN3/1 and shN3/2) or the control shRNA. In all samples analyzed, density values were normalized to the ratio measured in the control sample, which was set at 1. Mid panel: following shN3/1 transduction of DND41 cells, Notch3, MKP-1, p38, P-p38 and a-tubulin levels were analyzed by immunoblotting, and quantification was performed by P-p38 AlphaScreen SureFire kit (right panel), as described in section ‘Materials and methods’. Results are expressed as mean±s.d. of two independent experiments; *Po0.05.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Expressing, Solvent, Western Blot, In Vitro, Transfection, Plasmid Preparation, Control, shRNA, Transduction, Amplified Luminescent Proximity Homogenous Assay

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 4 MKP-1 modulates apoptosis of T-ALL cell lines. (a) Left panel: MKP-1 is expressed at higher levels in MOLT-3 primary cultures established from growing (GT) compared with dormant (DT) tumors by Western blot analysis. Right panel: Reduced apoptosis in primary cultures from GT compared with DT tumors following 6-h treatment with anysomicin (0.67 mg/ml). Apoptosis was evaluated by Annexin V staining and flow cytofluorimetric analysis. The columns report the mean values±s.d. of three independent experiments. *Po0.05 compared with untreated controls. (b) Transduction of MOLT-3 cells with lentiviral vectors encoding human MKP-1 (cytomegalovirus (CMV)–MKP-1) or shRNA targeting MKP-1 (shMKP-1) modulates MKP-1 expression levels in MOLT-3 cells, compared with controls (CMV; shRNA). The panels show the results of immunofluorescence analysis of MKP-1 expression in MOLT-3 cells transduced by the indicated constructs. Magnification, 200. (c) Detection of total and phosphorylated levels of JNK1-2 and p38 by Western blot analysis of lysates from MOLT-3 cells bearing modulated MKP-1 levels, treated or not with anisomycin. Membranes were probed with anti-b-actin as a loading control. One representative experiment of three performed is shown. (d) Measurements of apoptosis by Annexin V staining of T-ALL cells bearing increased (left panel) or reduced MKP-1 levels (right panels). MOLT-3 cells were treated for 6 h with anysomicin, 24 h with cisplatin (6 mg/ml), or 48-h serum starvation (fetal calf serum (FCS)) before staining with Annexin V–fluorescein isothiocyanate (FITC) and cytofluorimetric analysis. The columns report the mean values±s.d. of four independent experiments. *Po0.05 compared with controls. (e) Measurements of apoptosis of T-ALL cells bearing reduced MKP-1 levels following treatment with chemotherapeutics. Cell lines were treated for 24 h (MOLT-3) or 48 h (DND41) with cytarabine (20 nM), vincristine (10 mg/ml) or methotrexate (MTX, 300 nM) before staining with Annexin V–FITC and cytofluorimetric analysis. The columns report the mean values±s.d. of four independent experiments. *Po0.05 compared with controls.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Western Blot, Staining, Transduction, shRNA, Expressing, Construct, Control

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 5 MKP-1 attenuation modulates apoptosis and P-p38 levels in vivo. MOLT-3 or DND41 cells bearing normal or reduced MKP-1 levels were mixed with Matrigel and bFGF and injected s.c. into NOD/SCID mice (six animals per group). Before injection, cells were subjected to measurements of MKP-1 RNA levels, which indicated reduction of MKP-1 expression by 50% in shRNA targeting MKP-1 (shMKP-1)-transduced cells compared with shRNA controls (not shown). (a) Macroscopical analysis of s.c. MOLT-3 and DND41 tumors, 3-week after injection. (b) Measurements of apoptosis by Annexin V staining of MOLT-3 and DND41 tumors bearing normal or reduced MKP-1 levels. (c) Cytofluorimetric analysis of P-p38 levels in DND41 xenografts. The columns report the mean values±s.d. of 5–8 samples per group. *Po0.05 compared with controls.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: In Vivo, Injection, Expressing, shRNA, Staining

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 7 Expression of MKP-1 in primary T-ALL cells and correlation with the other molecular variables. (a) p38 phosphorylation in pediatric T-ALL samples inversely correlates with MKP-1 expression levels by western blot analysis. Membranes were probed with anti-b-actin as a loading control. (b) Notch 3 transcript levels by quantitative PCR in T-ALL samples characterized by low versus high MKP-1 levels. Results were normalized to the Notch3 transcript level in samples PDTALL6, which was set at 1. (c) Treatment of T-ALL cells with a g-secretase inhibitor (CompE) lowers MKP-1 protein expression. Primary cells were treated for 72 h with CompE (10 mM) or solvent (dimethylsulphoxide (DMSO)) before western blot analysis. Cells cultivated in vitro in the presence of DMSO were used as reference sample. Columns represent mean values±s.d. of the pooled experiments. *Po0.05.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Real-time Polymerase Chain Reaction, Solvent, In Vitro

Journal: Cancer Cell

Article Title: Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas

doi: 10.1016/j.ccell.2020.02.002

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were: p-SMAD1/5/8 (Cell Signaling #13820; 1:1000), p-SMAD1/5/8 (laboratory of Peter ten Dijke, 1:1000 ( )), SMAD1 (Cell Signaling #6944, 1:1000), p-SMAD2 (Cell Signaling #3108, 1:1000), SMAD2 (Cell Signaling #3103, 1:1000), Id1 (Santa Cruz #sc-133104, 1:1000), Id2 (Santa Cruz #sc-398104, 1:1000), Id3 (Santa Cruz #sc-56712), p-ERK1/2 (Cell Signaling #9101, 1:1000), ERK1/2 (Cell Signaling #9102, 1:1000), H3-K27me3 (Millipore #07-449, 1:1000), total H3 (Abcam #ab10799, 1:3000), Rb (Cell Signaling #9309, 1:1000), p-Rb (Cell Signaling #9308, 1:1000), β-Actin (Sigma #A2066, 1:5000), α-Tubulin (Sigma #T6199, 1:5000).

Techniques: Plasmid Preparation, Virus, Cloning, Recombinant, Cell Culture, Saline, Protease Inhibitor, Membrane, Western Blot, Blocking Assay, SYBR Green Assay, Transfection, Viscosity, Purification, cDNA Synthesis, Luminescence Assay, Reporter Assay, Flow Cytometry, RNA Sequencing, Real-time Polymerase Chain Reaction, Luciferase, Control, Software, Imaging

Journal: Cell reports

Article Title: Drp1 Promotes KRas-Driven Metabolic Changes to Drive Pancreatic Tumor Growth

doi: 10.1016/j.celrep.2019.07.031

Figure Lengend Snippet:

Article Snippet: p44/42 MAPK (Erk 1/2) , Cell Signaling Technology , CST-9102; RRID: AB_330744.

Techniques: Cell Viability Assay, CyQUANT Assay, Proliferation Assay, Luminescence Assay, Recombinant, Software

Journal: Journal of biomolecular screening

Article Title: Multidimensional profiling of CSF1R screening hits and inhibitors: assessing cellular activity, target residence time, and selectivity in a higher throughput way.

doi: 10.1177/1087057111418113

Figure Lengend Snippet: Fig. 2. Cellular assays for colony-stimulating factor 1 receptor (CSF1R). (A) GW-2580 effects on M-NFS-60 and NS0 cell proliferation. (B) Sorafenib effects on CSF1R and extracellular signal-regulated kinase (ERK) phosphorylation after CSF1 stimulation of THP1 cells. (C) AZD6244 effects in the same assays as panel B. The large difference in EC50 reflects that AZD-6244 is an MEK inhibitor. (D) Top panel: Western blot of lysed THP1 cells with an anti-phosphoERK1/2 antibody (phosphoERK1/2 bands are indicated by labels ERK1 and ERK2). Lane 1: no stimulus. Lane 2: CSF1 stimulus inhibited by GW-2580. Lane 3: CSF1 stimulus only. Lane 4: empty. Lane 5: marker. Bottom panel: control for total ERK. The ERK1 band (not indicated) is poorly visible just above the ERK2 band. Lanes in the bottom blot were digitally shuffled to match the lanes in the top blot.

Article Snippet: CSF1R and ERK target engagement assays Human monocytic tHp1 cells (atcc no. tiB-202) were stimulated with csf1, after which csf1r autophosphorylation and erk1/2 kinase activation were measured. tHp1 cells were cultured in Dulbecco’s modified eagle’s medium (Dmem)/f-12 medium (without phenol red) and supplemented with l-glutamine, penicillin, streptomycin, and 10% heat-inactivated fBs. in each well of a 96-well culture plate, 250 000 cells were dispensed (at 2·106 cells/ml) and rested overnight at 7 °c and 6% co2. the next day, compound was added to a final Dmso concentration of 0.1%, followed by 30 min preincubation and stimulation with 500 ng/ml of human csf1 for 5 min, after which the cells were put on ice. lysis buffer was added (from the perkinelmer [waltham, ma] alphascreen p-erk surefire kit), and cells were lysed for 10 min on a shaker. the lysates were frozen at –80 °c for further use. for detection of csf1r phosphorylation, a human phospho-csf1r duoset kit was used (Dyc3268; r&D systems, minneapolis, mn). maxisorb 96-well plates (nunc, roskilde, Denmark) were coated with mouse antihuman csf1r, blocked, and incubated with 100 μl of tHp1 cell lysate for 2 h at room temperature. after washing, the plates were incubated with horseradish peroxidase (Hrp)–labeled anti-ptyr. signal was detected by incubating with chromogen for 20 min and then reading absorbance at 450 nm on an enVision plate reader (perkinelmer). for detection of phosphorylated erk1/2 levels, 12.5 μl lysate was thawed out and mixed in a 384-well optiplate (perkinelmer) with 12.5 μl of surefire perk1/2 (cat. no. tgres10k; perkinelmer) bead mix that was prepared according to the manufacturer’s instructions. the mixture was shaken and placed in the dark for 2 h at room temperature. the fluorescent signal, which results from the proximity of two labeled antibodies against erk1/2 and phoshorylated erk1/2(pthr202/ tyr204) and diffusion of activated oxygen between them, was quantified in an enVision plate reader. for checking specificity of the signal, we ran western blots in a standard fashion. for primary antibodies, we used antiphosphorylated erk1/2 (cat. no. 4377; cell signaling, Danvers, ma) or anti-erk1/2 (cat. no. 700012; life technologies); for secondary antibody, we used goat-anti-rabbit Hrp (cat. no. w4011; promega, madison, wi). signal was generated using ecl mix (pierce, rockford, il) and captured on a gel imaging system (Fig. 2).

Techniques: Phospho-proteomics, Western Blot, Marker, Control

Journal: eLife

Article Title: Modulation of dopamine D 1 receptors via histamine H 3 receptors is a novel therapeutic target for Huntington's disease

doi: 10.7554/eLife.51093

Figure Lengend Snippet: STHdh Q7 ( A and C ) and STHdh Q111 ( B and D ) cells were time-dependent stimulated with 1 μM or 30 μM SKF 81297 and intracellular calcium release ( A and B ) or p-p38 phosphorylation ( C and D ) was determined. In ( A and B ), curves are mean ± SEM of 3 to 6 independent experiments. In ( C and D ) values represent mean ± SEM of two independent experiments performed per triplicate of percentage of phosphorylation respect to vehicle-treated cells with example gels shown of STHdh Q7 and STHdh Q111 treated with 30 μM SKF 81297 below. Student’s t test showed a significant (*p<0.05, **p<0.01, ***p<0.001) effect over not treated cells.

Article Snippet: Antibody , anti-phospho-p38 MAPK (Thr180/Tyr182) (rabbit polyclonal) , Cell Signaling , Cat. #9211S RRID: AB_331641 , Dilution: 1/1,000.

Techniques: Phospho-proteomics

Journal: eLife

Article Title: Modulation of dopamine D 1 receptors via histamine H 3 receptors is a novel therapeutic target for Huntington's disease

doi: 10.7554/eLife.51093

Figure Lengend Snippet: In ( A and B ), STHdh Q7 ( A ) or STHdh Q111 ( B ) cells were pre-treated for 20 min with vehicle or with the H 3 R antagonist thioperamide (10 μM) and were not stimulated or overstimulated with SKF 81297 (30 μM) prior intracellular calcium release determination. For each curve values are expressed as a percentage of increase with respect to untreated not overstimulated cells and are mean ± SEM of 3 to 9 independent experiments. In ( C ), STHdh Q7 or STHdh Q111 cells were treated for 20 min with medium (control), with SB 203580 (10 μM) or with the H 3 R antagonist thioperamide (10 μM). Cells were overstimulated with SKF 81297 (30 μM) and p38 phosphorylation was determined. Values represent mean ± SEM (n = 3) and are expressed as percentage over control. One-way ANOVA followed by Bonferroni post hoc tests showed a significant effect over control (**p<0.01, ***p<0.001) or over SKF 81297 treatment ( # p<0.05, ## p<0.01, ### p<0.001). In ( D ), cell viability was determined in STHdh Q7 (black curves) or STHdh Q111 cells (red curves) pre-treated for 60 min with the p38 inhibitor SB 203580 prior overstimulation with SKF 81297 (30 μM). Values represent mean ± SEM (n = 24 to 30) of the cell viability recovery expressed as in-fold respect to SKF 81297 treated cells ( D ).

Article Snippet: Antibody , anti-phospho-p38 MAPK (Thr180/Tyr182) (rabbit polyclonal) , Cell Signaling , Cat. #9211S RRID: AB_331641 , Dilution: 1/1,000.

Techniques: Control, Phospho-proteomics

Journal: eLife

Article Title: Modulation of dopamine D 1 receptors via histamine H 3 receptors is a novel therapeutic target for Huntington's disease

doi: 10.7554/eLife.51093

Figure Lengend Snippet:

Article Snippet: Antibody , anti-phospho-p38 MAPK (Thr180/Tyr182) (rabbit polyclonal) , Cell Signaling , Cat. #9211S RRID: AB_331641 , Dilution: 1/1,000.

Techniques: Diagnostic Assay, Recombinant, Control, Sequencing, In Situ, Reverse Transcription, Amplification, Amplified Luminescent Proximity Homogenous Assay, Software

Journal: iScience

Article Title: A small-molecule degrader selectively inhibits the growth of ALK -rearranged lung cancer with ceritinib resistance

doi: 10.1016/j.isci.2024.109015

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p44/42 ERK1/2 (Thr202/Tyr204) , Cell Signaling Technology , Cat# 4370; RRID: AB_2315112.

Techniques: Control, Virus, Recombinant, Expressing, Amplified Luminescent Proximity Homogenous Assay, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, Gel Extraction, Cloning, Caspase-Glo Assay, Cell Viability Assay, Staining, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Western Blot, Software, CRISPR

Journal: Cell Reports Medicine

Article Title: Combined inhibition of focal adhesion kinase and RAF/MEK elicits synergistic inhibition of melanoma growth and reduces metastases

doi: 10.1016/j.xcrm.2025.101943

Figure Lengend Snippet: Combined inhibition of RAF, MEK, and FAK abrogates melanoma cell growth in vitro (A and B) Cell proliferation was measured in YUMM3.2;Pten −/− ;Akt1 E17K cells treated in triplicate with increasing concentrations of VS-4718 (blue) or avutometinib (red). A Dunnett’s multiple comparison test was used to determine statistical significance (72 h). Mean values +/− SEM are reported. p values are as follows: p < 0.05 (∗), p < 0.001 (∗∗∗). (C) IC50 values of VS-4718- and avutometinib-treated cells. (D) Drug synergy was measured in duplicate using the Chou-Talalay combination index (CI) for increasing concentrations of VS-4718 and avutometinib in combination; values less than 1 denote synergy. (E and F) Immunoblotting was used to quantify levels of total FAK and P-FAK (Y397) in cells treated with increasing concentrations of VS-4718 and total ERK and P-ERK1/2 (T202/204) in cells treated with increasing concentrations of avutometinib for 2 h. P-FAK was normalized to total FAK, and P-ERK1/2 was normalized to total ERK. Histogram values are expressed as fold change relative to DMSO control. (G and H) Immunoblotting was used to quantify levels of total FAK and P-FAK (Y397) in cells treated with 600 nM of VS-4718 and total ERK and P-ERK1/2 (T202/204) in cells treated with 600 nM of avutometinib over 24 h. P-FAK was normalized to total FAK, and P-ERK1/2 was normalized to total ERK. Histogram values are expressed as fold change relative to DMSO control. See also Figure S1 and .

Article Snippet: Rabbit anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Inhibition, In Vitro, Comparison, Western Blot, Control

Journal: Cell Reports Medicine

Article Title: Combined inhibition of focal adhesion kinase and RAF/MEK elicits synergistic inhibition of melanoma growth and reduces metastases

doi: 10.1016/j.xcrm.2025.101943

Figure Lengend Snippet: Combined inhibition of RAF, MEK, and FAK increases melanoma cell death in vitro Cell viability was measured in YUMM3.2;Pten −/− ;AKT1 E17K cells treated in replicates of eight with 600 nM of encorafenib, avutometinib, and VS-4718 or a combination of each drug after 72 h. A one-way ANOVA was used to determine statistical significance. Mean values +/− SD are reported. p values are as follows: p < 0.01 (∗∗), p < 0.0001 (∗∗∗∗); ns, not significant. Immunoblotting was used to quantify levels of P-FAK (Y397, Y925), total FAK, P-AKT (S473, T308), total AKT, P-ERK1/2 (T202/204), total ERK1/2, cleaved caspase-3, and GAPDH (loading control).

Article Snippet: Rabbit anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Inhibition, In Vitro, Western Blot, Control

Journal: Cell Reports Medicine

Article Title: Combined inhibition of focal adhesion kinase and RAF/MEK elicits synergistic inhibition of melanoma growth and reduces metastases

doi: 10.1016/j.xcrm.2025.101943

Figure Lengend Snippet: Combined FAK/RAF/MEK inhibition significantly reduces tumor growth, prolongs overall survival, and reduces metastases in mice with established primary melanoma tumors (A) Schematic of subcutaneous tumor induction, BLI, and drug treatment in the syngeneic melanoma mouse model. Created with BioRender.com . (B) Mean tumor volume over time. C57BL/6 glowing head mice were transplanted subcutaneously with luciferase-expressing YUMM3.2;Pten −/− ;AKT1 E17K cells. Once tumors were measurable, mice were randomized into vehicle (black), VS-4718 (50 mg/kg PO BID; blue), avutometinib (0.3 mg/kg PO QD; red), or VS-4718 and avutometinib (purple) cohorts. A one-way ANOVA was used to determine statistical significance in tumor volume on day 11. Mean values +/− SEM are reported. Mouse numbers in each cohort are indicated in the legend. p values are as follows: p < 0.05 (∗), p < 0.01 (∗∗), p < 0.0001 (∗∗∗∗). (C) Waterfall plot depicting the tumor objective response rate from (B) at day 11. Each bar represents an individual mouse. (D) Kaplan-Meier percent survival curves for mice treated with vehicle (black), VS-4718 (blue), avutometinib (red), or VS-4718 and avutometinib (purple). A log rank (Mantel Cox) test was used to determine statistical significance. Mouse numbers in each cohort are indicated in the legend. p values are as follows: p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗); ns, not significant. (E) Bioluminescence imaging (BLI) on the lungs and brains of mice in (D) was performed ex vivo , following the injection of luciferin. A Tukey’s multiple comparison test was used to determine statistical significance in BLI radiance (photons/second/cm 2 /steradian [sr]). p value is as follows: p < 0.05 (∗). (F) Immunoblot analyses were performed using primary tumor lysates and antibodies against P-FAK (Y397), FAK, P-MEK1/2 (S217/221), MEK1/2, and GAPDH (loading control). (G) Quantitation of immunoblot data in (F) performed in duplicate. P-FAK was normalized to total FAK, P-MEK1/2 was normalized to total MEK1/2, and P-ERK1/2 was normalized to total ERK1/2. Values are expressed relative to vehicle control. p values are as follows: p < 0.05 (∗), p < 0.01 (∗∗). Total mouse numbers and sex of each are as follows: vehicle n = 11 (4 females and 7 males), VS-4718 n = 14 (7 females and 7 males), avutometinib n = 9 (3 females and 6 males), and VS-4718 and avutometinib n = 10 (5 females and 5 males). The number of mice differs across panels, as not all subjects were evaluable in each experimental condition. See also Figure S3 .

Article Snippet: Rabbit anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Inhibition, Luciferase, Expressing, Imaging, Ex Vivo, Injection, Comparison, Western Blot, Control, Quantitation Assay

Journal: Cell Reports Medicine

Article Title: Combined inhibition of focal adhesion kinase and RAF/MEK elicits synergistic inhibition of melanoma growth and reduces metastases

doi: 10.1016/j.xcrm.2025.101943

Figure Lengend Snippet: Combined FAK/RAF/MEK inhibition significantly prolongs survival in mice with existing brain metastases (A) Schematic of intracranial tumor induction, BLI, and drug treatment in the C57BL/6 syngeneic mouse model. Created with BioRender.com . (B) Kaplan-Meier percent survival curves for newborn glowing head mice intracranially injected with luciferase-expressing YUMM3.2;Pten −/− ;AKT1 E17K cells and treated with vehicle (black), VS-4718 (50 mg/kg PO BID; blue), avutometinib (0.3 mg/kg PO QD; red), or VS-4718 and avutometinib (purple) for 28 days upon weaning. A log rank (Mantel Cox) test was used to determine statistical differences. Mouse numbers in each cohort are indicated in the legend. p values are as follows: p < 0.05 (∗), p < 0.01 (∗∗). (C) Histological analyses were performed on the brain sections of mice. Sections were stained for H&E, and immunohistochemistry performed using antibodies against Ki67, P-FAK, P-ERK1/2, and HA (AKT1 E17K ); representative images of tumor regions are shown (left to right); scale bar, 50 μm. (D) Ki67, P-ERK, and P-FAK quantification in tumors. Three high power fields were evaluated for each sample. An unpaired t test was used to determine statistical significance. p values are as follows: p < 0.05 (∗), p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗). Total mouse numbers and sex of each are as follows: vehicle n = 6 (3 females and 3 males), VS-4718 n = 6 (1 female and 5 males), avutometinib n = 9 (5 females and 4 males), and VS-4718 and avutometinib n = 6 (1 female and 5 males). See also Figure S4 .

Article Snippet: Rabbit anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Inhibition, Injection, Luciferase, Expressing, Staining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Combined inhibition of focal adhesion kinase and RAF/MEK elicits synergistic inhibition of melanoma growth and reduces metastases

doi: 10.1016/j.xcrm.2025.101943

Figure Lengend Snippet:

Article Snippet: Rabbit anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Virus, Recombinant, Luminescence Assay, Luciferase, Software

Journal: Cell reports

Article Title: The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways

doi: 10.1016/j.celrep.2019.02.052

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-phospho-ERK1 and ERK2 (T202/Y204) , Cell Signaling Technology , Cat# 4376; RRID:AB_331772.

Techniques: Recombinant, Luminescence Assay, Mutagenesis, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Fusion Toxin BLyS-Gelonin Inhibits Growth of Malignant Human B Cell Lines In Vitro and In Vivo

doi: 10.1371/journal.pone.0047361

Figure Lengend Snippet: A. Cells were treated with BLyS-gel at 500 pM for 4, 8 or 24 hrs prior to collection of whole cell lysates for western blot analysis. Cells were also treated with anisomycin (ani) at 1 µg/ml for 1 hr as a positive control for RSR induction. Blots were probed using antibodies specific for phosphorylated and non-phosphorylated forms of p38 and JNK. Cleaved PARP was probed as a measure of cell death and α-tubulin was probed as a loading control. B. Cells were treated with BLyS-gel at 500 pM for 24 hrs or ani for 1 hr prior to collection of whole cell lysates. p38 phosphorylation was quantified using a quantitative ELISA assay. C. Cells were treated with a titer of the p38/JNK inhibitor SB203580 in the presence (gray bars) or absence (black bars) of BLyS-gel at 500 pM. Cell viability was analyzed following 72 hrs of treatment. D. Rec-1 cells were treated with SB203580 (SB) at 12.5 µM, BLyS-gel (Bg) at 500 pM, or the combination for 72 hrs prior to the collection of whole cell lysates for western blot analysis. Blots were probed as described in A. NT, no treatment.

Article Snippet: Ten microliters of cell lysates were transferred to wells of an opaque white 96-well 1⁄2 area polystyrene plates (Corning, Acton, MA) for quantitation of p38 phosphorylation using the AlphaScreen SureFire p38 MAPK (p-Thr180/Tyr182) assay kit (Perkin-Elmer) according to manufacturer's protocol.

Techniques: Western Blot, Positive Control, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay