Anti alk antibody

The following primary antibodies were used; anti‐FLAG antibody (mouse, M2; Sigma‐Aldrich; dilution used in WB, 1:1000), anti‐HA antibody (rat, #11867423001; Roche Applied Science; dilution used in immunocytochemistry (ICC): 1:1000), anti‐SMYD2 antibody (rabbit, D14H7; Cell Signaling Technology; dilution used in WB, 1:1000), anti‐ALK antibody (rabbit, D5F3; Cell Signaling Technology, Danvers, MA, USA; dilution used in WB, 1:1000), anti‐phospho‐Y1604‐ALK antibody (rabbit; Cell Signaling Technology; dilution used in WB: 1:1000), anti‐phospho‐Y1278/1282/1283‐ALK antibody (rabbit; Cell Signaling Technology; dilution used in WB, 1:1000), anti‐phospho‐S473‐AKT antibody (rabbit; Cell Signaling Technology; dilution used in WB, 1:500), and anti‐β‐actin antibody (mouse, 8H10D10; Cell Signaling Technology; dilution used in WB, 1:1000).

Cell viability was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Invitrogen). Absorbance was quantified with a ThermoFisher Spectrophotometer 1000 (Molecular Devices, Inc.) at a wavelength of 540 nm. Crizotinib (PF-02341066, Selleck Chemicals) (0.25 µM and 2 µM for H3122 and H2228 cells respectively) was used as a positive control.
Cells were lysed in buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid sodium salt, 0.1% SDS, 25 mM Tris-HCL (pH 7.4), and protease inhibitor cocktail set III (Wako Pure Chemical Industries, Japan). Proteins were separated by 9% and 11% SDS-PAGE for blotting with ALK and β-actin antibodies, respectively, and then transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Proteins were detected by immunoblotting using ECL Select detection reagent (GE Healthcare, Italy). Anti-ALK antibody (Cell Signaling Technology) was used at a dilution of 1:1000. Anti-β–actin antibody (Proteintech) was used at a dilution of 1:2000.