Journal: Oncoimmunology

Article Title: Melanomas prevent endothelial cell death under restrictive culture conditions by signaling through AKT and p38 MAPK/ ERK-1/2 cascades

doi: 10.1080/2162402X.2016.1219826

Figure Lengend Snippet: Melanoma conditioned medium induces a pro-survival signal transduction cascade (a) Caspase induction in endothelial cells (ECs) under hypoxia. ECs were treated with < 1 kDa fractions of various serum-free melanoma conditioned media (SF-CM), under hypoxic conditions. ECs cultured with HUVEC medium under normoxic conditions were used as a negative control for apoptosis and the addition of Etoposide (25 µg/mL) served as the positive control for apoptosis induction. Levels of active caspase 3/7 were measured at 16 h of treatment. Data are represented as mean ± SD of three independent experiments, conducted in triplicate. (b) Serum-starved ECs were treated with unfractionated or fractionated (< 1 kDa) SF-CM under hypoxic conditions. Cells were lysed and western blotting was performed to detect total and activated forms of AKT (MW, 60 kDa), ERK-1/2 (MW, 42.44 kDa), and p38 MAPK (MW, 43 kDa). SF-CM treatment lanes: 1. BLM, 2. Mel57, 3. 1F6, 4. PANC-1, 5. MCF-7, 6. hMEL, 7. Basal medium.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% nonfat dried milk (BioRad) in PBS/ 0.05% Tween-20, followed by overnight incubations with primary antibodies against total AKT (rabbit polyclonal, 1:1000 dilution), phospho-AKT (Ser473) (mouse monoclonal, 1:1000 dilution), total ERK-1/2 (rabbit polyclonal, 1:1000 dilution), phospho-ERK (mouse monoclonal, 1:1000 dilution), total p38 MAPK (rabbit polyclonal, 1:1000 dilution), and Phospho-p38 MAPK (Thr180/Tyr182) (mouse monoclonal, 1:2000 dilution).

Techniques: Transduction, Cell Culture, Negative Control, Positive Control, Western Blot

Journal: Journal of Dental Sciences

Article Title: Overexpression of sprouty 1 protein in human oral squamous cell carcinogenesis

doi: 10.1016/j.jds.2020.07.013

Figure Lengend Snippet: Western blot analyses: phosphor-ERK and total-ERK in human oral cancer cell lines as compared with a primary culture of normal oral mucosa (HOK). Upregulation of phosphor-ERK (normalized to total-ERK) in OECM1 and Ca922 cell lines as compared with HOK, and a slightly decreased expression in SAS as compared with HOK. Results were quantified using densitometric analysis, normalized to the level of β-actin, and expressed as a fold change relative to the normal oral mucosa. Bars represent means ± standard deviation of the mean (∗ P < 0.05). A representative result of three independent experiments is shown.

Article Snippet: Further, samples were analyzed using 10% SDS-PAGE (Sigma-Aldrich) gels, and the proteins were transmitted onto a PVDF membrane (Sigma-Aldrich) using Bio-Rad's transblot with primary antibodies against phosphor-ERK (Boster Biological Technology, CA, USA; Cat. No. P00104; 1:1000) and total-ERK (Boster Biological Technology; Cat. No. P00104; 1:1000), with species specificity for human tissues and an observed molecular weight of 42–44 kDa; and β-actin (Sigma-Aldrich; 1:1000), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich; 1:5000).

Techniques: Western Blot, Expressing, Standard Deviation