Erk sc 93

Cancer cell lines were purchased from ATCC, Manassas, VA. Human Mammary Epithelial cells immortalized by telomerase expression (tert-HMEC) have been described previously [35 (link)]. Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum. Cell extracts were prepared as described previously [36 (link)] and immunoblot analysis was carried out with the following antibodies: EGFR (#4267), phospho-EGFR[Y845] (#6963), PARP (#9532), phospho-Akt[T308] (#9275), Akt (#4691), phospho-Erk (#9101), HER2 (#2165), HER3 (#4754), phospho-HER2[Y877] (#2241), and phospho-S6 (#2211) from Cell Signaling Technology, Danvers, MA; Erk (sc-93), phospho-Tyrosine (sc-7020), and Actin (sc-1616) from Santa Cruz Biotechnology, Santa Cruz, CA; phospho-Tyrosine (4G-10, 05–321) from Millipore, Temecula, CA; E-cadherin (610182) from BD Transduction Laboratories, San Jose, CA.

Proteins extracted in RIPA buffer (50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) supplemented with NaF, NaVO₄, and a protease inhibitor cocktail (Sigma-Aldrich, P2714) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blotted with antibodies against human LC3 (#2775); phospho-AMPKα (Thr172, #2535); AMPKα (#2603) (from Cell Signaling Technology, Beverly, USA); Erk (SC-93); phospho-ERK (Tyr204 and SC-7383); ERK1 (SC-376852); p62 sequestosome (SC-25575); Lamp1 (SC-20011); Lamp2 (SC-5571); Limp2 (SC-55570) (from Santa Cruz Biotechnology, Dallas, TX, USA); and β-actin (A5441, Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies, and protein bands were visualized using SuperSignal West Femto substrate (Thermo Fisher, Waltham, MA, USA).

Antibodies to hnRNPA1 (ab5832, ab50492), hnRNPA3 (ab50949), hnRNPA₂/B₁ (ab64800), hnRNPL (ab6106, ab65049) and GAPDH (ab8245) or β-actin (ab8227) as loading controls, were from Abcam. Antibodies against acetyl-Lys (9441), pAKT (40665) and AKT (9272) were from Cell Signaling Technology. Other antibodies used were: ERK (sc-93) and pERK (sc-7383) from Santa Cruz Biotechnology; KRAS (05–516) from Millipore and Talin (T3287) from Sigma-Aldrich.
Epidermal growth factor (EGF 20ng/ml), trichostatin (TSA, 0.5μM) and sodium butyrate were from Sigma-Aldrich, UO126 (10μM) from Promega, LY294002 (10μM) from Calbiochem and Fibronectin from BD Biosciences.