Anti fgfr4

Freshly cultured cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Fixed cells were washed and permeabilized in PBS supplemented with 0.1% Triton X‐100 or 0.1% saponin (#30502‐42, Nacalai Tesque, Japan). Blocking was done in PBST including 1% bovine serum albumin for 30 min at room temperature. The following antibodies were employed for immunofluorescence: anti‐Crb3a (2 μg/mL), anti‐FGFR1 (#9740, Cell Signaling Technology) and anti‐FGFR4 (1:100 dilution). Antibodies were diluted in PBS and immunoreactions were carried out overnight at 4°C in a humid chamber. The fluorescent dye‐conjugated secondary antibody reaction was performed for 1 hr at room temperature. Counterstain with Rhodamine‐Phalloidin (1:1,000 dilution, #PHDR1, Cytoskeleton, USA) and/or Hoechst 33342 was done simultaneously with the secondary antibody reaction. The fluorescent image was acquired with a fluorescent inverted microscope (IX71, Olympus) or confocal microscope (LSM 510 Meta, Zeiss, Germany).

Cell lysates were prepared in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein lysates (25–35 μg/lane), as determined by BCA protein assay (Life Technologies), were then separated by SDS-PAGE (NuPAGE; 4%–12% Bis-Tris gels; Invitrogen) and transferred on to a nitrocellulose membrane that was probed with anti-FGFR4 (Cell Signaling Technology, cat. #8562), anti-IGF-1Rβ (Cell Signaling Technology, cat. #9750), anti-MYOD1 (Cell Signaling Technology, cat. # 13812), anti-FOXO1 (Cell Signaling Technology, cat. #2880) and anti-β-Actin (Cell Signaling Technology, cat. #4967) primary antibodies followed by anti-rabbit IgG, HRP-linked secondary antibody (Cell Signaling Technology, cat. #7074) before detection using an iBright CL1000 imaging system (Thermo Fisher Scientific, MA, USA). iBright analysis software was used for quantification of the intensity of bands of interest.