Fgfr4

The human CCA tissue microarray was purchased from Pantomics (LVC1261). Following antigen retrieval, the tissue slides were incubated with primary antibodies against METTL16 (Abclonal, A15894), PRDM15 (Thermo Fisher Scientific, PA5-55,036, RRID: AB_2645923), and FGFR4 (Proteintech, 11,098–1-AP). After rinsing with TBS, the slide was incubated for 1 h at room temperature with a horseradish peroxidase-conjugated second antibody. After washing with TBS, the slide was incubated for 5 min at room temperature with 3,3′-diaminobenzidine (DAB) for chromogenic development. The IHC score was determined by multiplying the staining intensity (negative: 0; mild: 1; moderate: 2; severe: 3) with the staining area (negative: 0; ≤ 30%: 1; > 30% and ≤ 60%: 2; > 60%: 3).

Total protein extracts were isolated with Radioimmunoprecipitation assay (RIPA) buffer in the presence of protease and phosphatase inhibitor cocktail (Roche). Equal quantities of protein were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, immunoblotted with the appropriate antibodies, and revealed using Odyssey. The antibodies used in this study were as follows: METTL16 (Cell Signaling Technology, #87,538), PRDM15 (Proteintech, 25,590–1-AP), SETD5 (Abclonal, A7304, RRID:AB_2767845), KMT2D (Proteintech, 27,266–1-AP), NSD2 (Abclonal, A7938, RRID:AB_2772896), FGFR4 (Proteintech, 67,800–1-Ig), YTHDF1 (Proteintech, 17,479–1-AP, RRID:AB_2217473), YTHDF3 (Cell Signaling Technology, #24,206), FLAG (Cell Signaling Technology, #14,793), HA (Cell Signaling Technology, #3724), H3K27ac (Cell Signaling Technology, #8173), p300 (Cell Signaling Technology, #54,062), YY1 (Cell Signaling Technology, #46395S), β-actin (Sigma, A2228), and the anti-rabbit and anti-mouse secondary antibodies (LI-COR, Ride® 800CW).