α enolase

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).

Mouse monoclonal antibodies (mAbs) recognizing triosephosphate isomerase (TPIS, catalog reference TPI, H-11), glyceraldehyde phosphate dehydrogenase (GAPDH, catalog referenceA-3), β-actin (ACTB, catalog reference ACTBD11B7), α-enolase (ENOA, catalog reference A-5), heat shock 70 kDa protein (HSP71, catalog reference 3A3) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used as a benchmark on the 2D immunoblots, providing anchors for the inter-assay alignment by generating a landmark map. Their localization on different proteomic maps has been extensively described (http://www.uniprot.org). The selection of these benchmarks was based on both their large pI and MW distributions on the proteomic map.