Pan actin

Cerebella were lysed in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease cocktail inhibitor (Sigma). Fifty micrograms of proteins were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were then transferred to Polyvinylidene fluoride membranes (GE Healthcare, Life science). The following primary antibodies were diluted in 5% milk in TBS containing 0.1% Tween‐20: RIPK3 (Cell Signaling, 15828S; 1:1,000), Pan‐Actin (Millipore, MAB1501R, 1:4,000).

Antibodies were obtained from the following commercial sources: PARP, caspase-3, caspase-8, caspase-9, phospho-Erk, and Erk (Cell Signaling Technology); p-c-Met, c-Met, and FGFR3 (Santa Cruz Biotechnology); pan-actin (Millipore); phospho-stat3-Ser⁷²⁷ and Stat3 (BD Biosciences). propidium iodide, MTT, z-VAD–FMK, PD98059, PD173074, and all of the other chemical reagents were obtained from Sigma. RPMI-1640 medium, Dulbecco's Modified Eagle Medium (DMEM), FBS, penicillin, streptomycin, and all other tissue culture regents were obtained from GIBCO/BRL Life Technologies. Sorafenib (purity ≥ 99%) was purchased from Biovision. Tivantinib (ARQ 197) was purchased from ChemieTek. For in vitro administration, Sorafenib, tivantinib or DE605 (structure and scheme of DE605 synthesis shown in supplemental Fig. S1) were dissolved in DMSO (Sigma) to a concentration of 10 mM and further diluted to appropriate final concentration in RPMI 1640 with 10% fetal bovine serum. DMSO in the final solution did not exceed 0.1% (v/v). For in vivo testing, Sorafenib or DE605 were dissolved in Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethanol) at 4 × concentration. This 4 × solution was prepared fresh every 4 days. Final dosing concentration was prepared by diluting the 4 × solution to 1× with sterile water. The 1× solution was prepared just before it was given to the mice.