pathscan phospho src tyr416 sandwich elisa kit  (Cell Signaling Technology Inc)

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA activates Src kinase in A549 cells, but reduces the activity of recombinant Src kinase in a cell-free assay. (A) A549 cells were treated with AcoA for 1 to 24 h, lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, ** p < 0.01, *** p < 0.001 in comparison to cells incubated with vehicle for the indicated time. (B) AcoA, ouabain and digoxin reduce Src kinase activity in a cell-free assay. Enzymatic activity of recombinant c-Src kinase (0.1 U/mL) pretreated with AcoA, ouabain, digoxin, doxorubicin, or the positive control staurosporine (all 100 nM) for 30 min, was analyzed by a kinase assay. Data are mean ± SD of triplicates, * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to the vehicle control. (C) Src activation ( p -Src Y416 expression) in the NCI-60 tumor cell lines does not correlate with AcoA cytotoxicity (GI 50 values) in the same cell lines (original data were obtained from NCI database). Red dot indicates A549 cell line. Pearson correlation test. (D) AcoA and ouabain-induced cell toxicity is unaffected by the Src inhibitor PP2. Cell viability of A549 cells pretreated for 4 h with the Src kinase family inhibitor PP2 and incubated for 48 h with AcoA or ouabain (100 nM). XTT assay, mean ± SEM of N = 3.

Article Snippet: To measure Src kinase phosphorlyation, we used an ELISA Kit (PathScan Phospho-Src (Tyr416) Sandwich ELISA Kit, Cell Signaling Technology).

Techniques: Activity Assay, Recombinant, Cell-Free Assay, Activation Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Comparison, Incubation, Positive Control, Kinase Assay, Control, Expressing, XTT Assay

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA leads to endosomal EGFR arrest and inhibits EGF-induced degradation of EGFR (A) AcoA induces endosomal arrest in EGFR biosensor cells. After 1 h preincubation with monensin (1 µM), AcoA, digoxin, doxorubicin (all 100 nM), or vehicle, cells were stimulated with 100 ng/ml EGF and the formation of green fluorescent vesicles indicating activated and internalized EGFR was monitored microscopically. Representative images are shown. The graphs on the right show the number of fluorescent vesicles/cell at the respective time point. Data are mean ± SEM of N = 3 independent experiments, *** p < 0.001. (B) Quantification of cellular EGFR in A549 cells treated as in (A) using a commercial ELISA In-cell ELISA assay. The amount of EGFR was normalized to cell number assessed by crystal violet staining. Data are mean ± SEM of N = 3 independent experiments, * p < 0.05 vs. control, # p < 0.05 vs. EGF treatment group. (C) At the same conditions, EGFR activation was assessed using ELISA for the Y1173 phosphorylation site of EGFR. EGFR phosphorylation is expressed as the ratio of phosphorylated EGFR to total EGFR. Data are mean ± SEM of N = 3, * p < 0.05 vs. control. (D) Src kinase activation was measured as a downstream target of EGFR. A549 cells treated as in (A) were lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, * p < 0.05 vs. control, ** p < 0.01 vs. control, # p < 0.05 vs. EGF treatment group.

Article Snippet: To measure Src kinase phosphorlyation, we used an ELISA Kit (PathScan Phospho-Src (Tyr416) Sandwich ELISA Kit, Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Staining, Control, Activation Assay, Phospho-proteomics