Anti fgfr3

IHC analysis was employed to evaluate FGFR protein expression. Antibodies used were anti-FGFR1 (Cell Signaling Technology; 1:500), anti-FGFR2 (R & D Systems; 22 μg/mL), anti-FGFR3 (Cell Signaling Technology; 1:50), and anti-FGFR4 (R & D Systems; 15 μg/mL). PBS was used as negative control. Microscopically, ten high-power (40×) fields from each section were observed randomly and 100 cells were scored. Staining was semi-quantified into four scores based upon the number of positive cells: < 5% (score 0), 5–25% (score 1), 26–50% (score 2), 51–75% (score 3), and > 75% (score 4). Staining intensity followed a four-score classification: no cell stain (score 0), yellow (score 1), tan (score 2) and brown (score 3). Final IHC scores were calculated by addition of intensity and extent scores, and the results were divided as follows: negative (−; scores 0 and 1), weakly positive (+; scores 2 and 3), moderately positive (++; scores 3–5), and strongly positive (+++; scores 6 and 7). Results of staining for FGFR1–4 were organized into low expression (negative and weakly positive) and high expression (moderately and strongly positive) groups [28 (link)].

Immunoprecipitation analyses were performed using the Direct Magnetic IP/Co-IP Kit (ThermoFisher, 88828) according to the manual. Briefly, indicated antibodies were bound to the beads for 60 min. Cell lysates were incubated with antibody-bound beads overnight at 4 °C. Washed the beads twice with Wash Buffer and once with ultrapure water. Eluted bound antigen.
For Western blot analyses, the cells were digested in RIPA buffer with presence of Protease Inhibitor Cocktail (ThermoFisher, 87785). Protein concentration was quantified using the BCA Protein Assay Kit (ThermoFisher, 23227). After electrophoresis in SDS-PAGE, the proteins were transferred to PVDF membranes (Bio-Rad, 1620177). After blocking with PBS containing 5% nonfat milk, blots were immunoblotted with the indicated primary antibodies. The antibodies included anti-HA (Cell Signaling technology, #3724) and anti-myc (Cell Signaling technology, 2276), anti-FGFR3 (Cell Signaling technology, #4574), anti-PTEN (Cell Signaling Technology, #9599), anti-Akt (Cell Signaling Technology, #4691), anti-p-Akt (Cell Signaling Technology, #4060), anti-TET2 (Cell Signaling Technology, #18950) and anti-GAPDH (Cell Signaling technology, #5174).