P fgfr3

Cells were seeded into 6-well plates overnight and treated with the indicated compounds at various concentrations for the indicated times. Proteins were extracted using a lysis buffer from whole-cell lysates, were fractionated in SDS-PAGE gels, and were transferred onto polyvinylidene difluoride (PVDF) membrane for protein blot analysis.
The primary antibodies for detection of the indicated protein were obtained from the following sources: caspase 3, LC3, and GAPDH, which were purchased from Novus (Littleton, CO, USA), as well as caspase 8, caspase 9, PARP, γH2AX, AKT, p-AKT (T308), p-AKT (S473), STAT3, p-STAT3 (Y705), and ATG5, which were purchased from Cell signaling (Dallas, TX, USA). P62 was purchased from GeneTex (Irvine, CA, USA). FGFR3 and p-FGFR3 were purchased from Abcam (Cambridge, UK). DAPK2 was purchased from Arigo Biolaboratories (Hsinchu, Taiwan). DAPK2 protein expression was stably knocked down using shRNA expressing lentiviral particles against DAPK2 (#1, TRCN0000199968; #2, TRCN0000001718), and shRNA-targeting luciferase was used as a negative control (TRCN0000072249); both were purchased from National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). The cells were seeded into a 6-well plate for transduction. Stable knockdown cells were selected and maintained with puromycin (2 μg/mL).

Equal amounts of protein were separated using SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. After blocking with nonfatty milk, these membranes were incubated with primary antibodies as follows: pFGFR3 (Abcam, 1/1000); FGFR3 (Santa Cruz, 1/200); pAkt (CST, 1/1000); Akt (CST, 1/1000); pErk1/2 (CST, 1/2000); Erk1/2 (CST, 1/2000); Snail (CST, 1/1000); Vimentin (CST, 1/1000); E-Cadherin (CST, 1/1000); and β-actin (Boster, 1/2000). The membranes were then incubated with horseradish peroxidase-conjugated IgG (CST, 1/3000). Protein blot was detected using the ECL system (Millipore).

Total protein was lysed with proteinase inhibitor cocktail and radioimmunoprecipitation assay (RIPA) buffer with and conditionally adding phosphatase inhibitors (Sangon Biotech, Shanghai, China). The protein samples were then mixed with 5 × loading buffer, then denatured at 95℃ for 5 min, and electrotransferred to polyvinylidene fluoride (PVDF) membrane by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After blocking with 5% skim milk, the membrane was incubated in the primary antibody overnight at 4 °C, and then the secondary antibody diluted in Tris-buffered saline with Tween 20 (TBST) was used. Then, the protein-antibody complexes were detected using the enhanced chemiluminescence (ECL) on the Tanon 5200 Multi intelligent imaging system. The primary antibody used in this study are shown as below: VEGFR1 (Affinity, AF6204), VEGFR2 (Cell signaling Technology, 9698), VEGFR3 (Affinity, AF4201), FGFR1 (Signalway Antibody, 49,175), FGFR2 (Abcam, ab109372), FGFR3 (Abcam, ab133644), FGFR4 (Abcam, ab119378), c-Kit (Bioworld Technology, BS2433), PDGFRβ (Bioworld Technology, BS1764), GAPDH (Proteintech, 60,004), p-FGFR3 (Abcam, ab155960), AKT (Affinity, AF6261), p-AKT (Affinity, AF0016), mTOR (Affinity, AF6308), p-Mtor (Affinity, AF3308), BCL2 (Proteintech, 12,789–1-AP), BAX (Abcam, ab32503), METTL3 (Proteintech, 15,073–1-AP).