Immunoblotting and Signaling Pathway Analysis

Immunoblotting was carried out using 15 or 30 µg of total protein lysate as described previously. The following primary antibodies were used: anti-β-actin and anti-Laminin (Abcam, Cambridge, UK); anti-ERK1 K-23 and anti-FGFR3 B-9 (Santa Cruz Biotechnology); anti-Phospho ERK1/2, T202/Y204, anti-α-E-Catenin 23B2 and anti-Phospho-α-E-Catenin S652 (Cell Signalling, Hitchin, Hertfordshire, UK). Anti-mouse or anti-rabbit IgG HRP linked secondary antibodies (Cell Signalling, Hitchin, Hertfordshire, UK) were used.
The PathScan^® Intracellular Signalling Array Kit (Cell Signalling, Hitchin, Hertfordshire, UK) was used according to manufacturer’s instructions to explore the phosphorylation status of proteins fundamental to signal transduction. Slide images were captured using Odyssey Fc System (Li-Cor Biosciences).
Immunoprecipitation for both FGFR3 WT and FGFR3-TACC3 (RT112) fusion were performed as in [48 (link)]. Immunoprecipitation of Topoisomerase IIα used 100 ug of starting material and was precipitated using the anti-TOPO IIα antibody (Cell Signalling, Hitchin, Hertfordshire, UK). Eluted protein was probed via immunoblotting using TOPO IIα and pS 1469 TOPO IIα antibodies (Cell Signaling, Hitchin, Hertfordshire, UK). Additional information can be found in the Supplementary Materials and Methods.