Journal: Science translational medicine

Article Title: An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma

doi: 10.1126/scitranslmed.aau9732

Figure Lengend Snippet: Neuroblastoma cells expressing wild-type ALK (NB-1 and IMR-32) and mutated ALK (SK-N-SH) or with no detectable ALK expression (SK-N-AS) were treated with increasing doses of CDX-0125-TEI, a control IgG1 antibody conjugated with TEI (IgG1-TEI), or the free payload (NMS-P528). Titration of CDX-0125-TEI induced cytotoxic activity in all ALK-expressing models, with IC50 values in the picomolar range and independent of ALK mutation status or number of cell surface ALK receptors. By contrast, no measurable IC50 value could be derived in the ALK-negative cell line SK-N-AS. Free NMS-P528 elicited complete cell killing at sub-picomolar concentrations in all cell lines tested. Quoted IC50 and ALK expression values reflect calculated means and SEM (n = 3) from at least three independent experiments.

Article Snippet: Upon immobilization onto polyvinylidene difluoride (PVDF) membranes, proteins were labeled using one of the following antibodies: phosphorylated ALK (Y1278), total ALK, cleaved caspase-3, and GAPDH (Cell Signaling Technology).

Techniques: Expressing, Titration, Activity Assay, Mutagenesis, Derivative Assay

Journal: Science translational medicine

Article Title: An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma

doi: 10.1126/scitranslmed.aau9732

Figure Lengend Snippet: (A) Apoptosis was quantified by annexin V–FITC (fluorescein isothiocyanate) and analyzed by FACS (fluorescence-activated cell sorting) in neuroblastoma cells (SK-N-AS, COG-N-453, NBL-S, Felix, and NB-1) treated with CDX-0125, CDX-0125-TEI, NMS-P528, or IgG1 control at their predetermined IC50 values. Values from a representative experiment are shown. Experiments were performed three independent times. (B) NBL-S and (C) COG-N-453 were subjected to immunoblot analysis and probed for the apoptosis marker cleaved caspase-3 and GAPDH as a loading control. Each bracket represents three independent samples. WT, wild type.

Article Snippet: Upon immobilization onto polyvinylidene difluoride (PVDF) membranes, proteins were labeled using one of the following antibodies: phosphorylated ALK (Y1278), total ALK, cleaved caspase-3, and GAPDH (Cell Signaling Technology).

Techniques: Fluorescence, FACS, Western Blot, Marker

Journal: Genes, brain, and behavior

Article Title: Binge-Like Ethanol Drinking Activates ALK Signaling and Increases the Expression of STAT3 Target Genes in the Mouse Hippocampus and Prefrontal Cortex

doi: 10.1111/gbb.12729

Figure Lengend Snippet: Activation of ALK in mouse medial prefrontal cortex (PFC) and ventral hippocampus (HPC) after binge-like ethanol drinking. Mice drank ethanol or water in the DID protocol for 4 days and the prefrontal cortex (PFC, n = 12 – 13 per sex per group) and ventral hippocampus (HPC, n = 6 per sex per group) were collected immediately after the 4th drinking session. Protein lysates were subjected to western blotting with antibodies to phosphorylated ALK (pALK), ALK, phosphorylated STAT3 (pSTAT3), STAT3, and β-actin as a protein loading reference on the gels. (A) Representative western blots from PFC samples. M, male; F, female. (B-E, G-J) Quantification of relative band intensity from western blots of the PFC (B-E) and the HPC (G-J) for pALK (B, G), ALK (C, H), pSTAT3 (D, I), and STAT3 (E, J). (F, K) Correlations between pSTAT3 and pALK in the PFC (F) and HPC (K). The R2 and p-values indicate a significant association between pSTAT3 and pALK in mice that consumed ethanol. Data are shown as the mean ± S.E.M. *p<0.05 and **p< 0.01, main effect of ethanol by 2-way ANOVA.

Article Snippet: Primary antibodies were the following: STAT3, Cell Signaling Technology #9139, RRID: AB_331757, 1:1000 dilution; phosphorylated STAT3 (pSTAT3, Y705), Cell Signaling Technology #9145, RRID: AB_2491009, 1:1000 dilution; ALK #3815 18 , 1:500 dilution; phosphorylated ALK (pALK, Y1278), Cell Signaling Technology #6941, RRID: AB_10860598, 1:500 dilution; and β-actin, Sigma-Aldrich #A5441, RRID: AB_476744, 1:10,000 dilution.

Techniques: Activation Assay, Western Blot