Cell Signaling Pathways in Melanoma Cells

Cell lysates were harvested and processed as described in refs [14 (link), 29 (link), 31 (link), 34 (link)]. In brief, 1.0 × 10⁶ melanoma cells were seeded in 100 mm culture dishes, 48 hours later; cells were treated with inactive derivatives of abietic acid, 2a & 5c (30–50 μmol/L) or active derivatives of leelamine, 4a & 5a (3–5 μmol/L) for 24 hours. Protein lysates were collected for Western blotting analysis. Blots were probed with antibodies according to each supplier's recommendations: antibodies to IGF-1R, total AKT, phosphor AKT (Ser473), total PRAS40, phospho-PRAS40 (Thr246), total ERK1/2, phospho-ERK1/2 (Thr202/Tyr 204), total CDK2, phospho-CDK2 (Thr160), p62, LC3B, total STAT, phospho-STAT3 (Tyr705), caspase 3 and cleaved PARP from Cell Signaling Technology (Danvers, MA); cyclin D1, α-enolase and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoblots were developed using the enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Rockford, IL).

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Gowda R., Inamdar G.S., Kuzu O., Dinavahi S.S., Krzeminski J., Battu M.B., Voleti S.R., Amin S, & Robertson G.P. (2017). Identifying the structure-activity relationship of leelamine necessary for inhibiting intracellular cholesterol transport. Oncotarget, 8(17), 28260-28277.

Publication 2017

phospho-PRAS40 total ERK1/2 cleaved PARP cyclin D1 α-enolase with horseradish peroxidase ECL) detection system

Abietic acid Antibodies Caspase 3 Cdk2 Cells Chemiluminescence Cyclin d1 Derivatives Dishes Erk1 Horseradish peroxidase Igf 1r Immunoblots Leelamine Melanoma Phosphor Protein Stat3 Western blotting analysis α enolase

Corresponding Organization :

Other organizations : Pennsylvania State University, Penn State Milton S. Hershey Medical Center

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Variable analysis

independent variables

Inactive derivatives of abietic acid (2a & 5c) at 30–50 μmol/L
Active derivatives of leelamine (4a & 5a) at 3–5 μmol/L

dependent variables

Protein expression levels of IGF-1R, total AKT, phospho-AKT (Ser473), total PRAS40, phospho-PRAS40 (Thr246), total ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), total CDK2, phospho-CDK2 (Thr160), p62, LC3B, total STAT, phospho-STAT3 (Tyr705), caspase 3, cleaved PARP, cyclin D1, and α-enolase

control variables

Melanoma cell line used (1.0 × 10^6 cells seeded)
Cell culture conditions (100 mm culture dishes, 48-hour incubation prior to treatment)
Treatment duration (24 hours)

controls

No positive or negative controls are explicitly mentioned in the input protocol.

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