Cells were treated for 45 min with either JNJ42756493 or control-treated with the corresponding concentration of JNJ427756493 diluent DMSO and for the last 15 min with or without 10 ng/mL of recombinant FGF1 (PeproTech, #100–17A, Rocky Hill, NJ, USA). Treated and control cells were then washed with PBS and were extracted using a 1.5 X Laemmli lysis buffer. Whole cell proteins (30 μg) were electrophoresed on 8–10% SDS-PAGE gels and transferred to PVDF membranes (Immobilon, Millipore, Burlington, MA, USA). Membranes were blocked with 5% BSA7030 (Sigma-Aldrich), for phosphorylated FRS2 or low-fat milk, then incubated overnight at 4°C with the following antibodies: anti-phospho-ERK (Thr202/Tyr204, #4370), anti-ERK (137F5, #4695), anti-phospho-AKT (Ser473, #4060), anti-AKT (11E7, #4685), anti-FGFR1 (D8E4, #9740), anti-FGFR4 (D3B12, #8562), anti-FGFR2 (D4L2V, #23328), anti-FGFR3 (C51F2, #4574), and anti-phosphoFRS2α (Tyr436, #3861) from Cell Signaling Technologies, anti-FRS2 (ab183492, Abcam), anti-MDM2 (Ab1-IF2, OP46, Calbiochem, Darmstadt, Germany), anti-P53 (DO-1, sc-126, Santa Cruz Biotechnology), anti-P21WAF1 (Ab-1, EA-10, Millipore), anti-βtubulin (66240–1-Ig, ProteinTech, Rosemont, IL, USA), and anti-GAPDH (60004–1-Ig, ProteinTech).
Quantification of the WB was performed using ImageJ software. Images are representative of the results obtained in three separate experiments.
Quantification of the WB was performed using ImageJ software. Images are representative of the results obtained in three separate experiments.