Radioimmunoprecipitation assay (ripa)

SK-MEL-2 and G-361 cells were treated with various concentrations of 7,8-DHF for different incubation times (100, 200, and 300 µM, for 12 and 24 h) and washed twice with DPBS. Cells were homogenized well with a protein extraction solution (RIPA) (ELPIS Biotech Inc., Daejeon, Korea). The extracted proteins were quantified using the Pierce^® BCA Protein Assay kit (Thermo Fisher Scientific lnc., Waltham, MA, USA). Equal amounts of protein samples were separated by 8%, 10%, and 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) along with protein markers and then transferred to polyvinylidene difluoride (PVDF) blotting membranes. The membranes were blocked at room temperature for one hour with 5% non-fat dried milk in 1 × TBS (Biosesang, Seongnam, Gyeonggi, Korea) containing 0.1% Tween-20 (Bio-Rad Inc., CA, USA) and then incubated overnight at 4 ℃ with specific primary antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for two hours, immunoreactive bands were visualized using a SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific lnc., Waltham, MA, USA).

Cells were lysed in RIPA (radioimmunoprecipitation assay) buffer (Elpis Biotech, Daejeon, Korea) supplemented with a protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA) and protein phosphatase inhibitors (Calbiochem). Protein concentrations were determined using a BCA assay kit (Pierce, Rockford, IL, USA). Proteins (10 μg/sample) were resolved in an SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). Membranes were blocked with 5% skim milk prior to western blot analysis. Chemiluminescence was detected using an ECL kit (Advansta Corp., Menlo Park, CA, USA) and the Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK). The following primary antibodies were used on fresh individual membranes to minimize interference by stripping and reprobing: phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵), JNK, phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²), p38 MAPK, phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), ERK1/2, phospho-Src (Tyr⁴¹⁶), Src, phospho-Lyn (Tyr⁵⁰⁷), Lyn, phospho-Akt (Ser⁴⁷³), Akt, phospho-PI3K p85 (Tyr⁴⁵⁸/Tyr¹⁹⁹), PI3K p85, PI3K p110α, PI3K p110β, PI3K p110γ, PI3K p110δ, phospho-PTEN (Ser³⁸⁰/Thr^382/383), PTEN, and Fyn (Cell Signaling Technology, Beverly, MA, USA); and β-actin and phospho-Fyn (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All the raw data from immunoblotting experiments are presented in Figure S4.