Anti phospho fgfr1

Total RNA was isolated using Trizol (Invitrogen, Waltham, MA) and retro-transcribed with a QuantiTect reverse transcription kit (Qiagen, Valencia, CA). Amplification was performed using standard PCR (New England Bio, MA) in combination with specific primers and conditions (available upon request). Proteins were isolated as described previously.^{8 (link)} Whole-cell lysates (30 µg) were separated using SDS-PAGE and immunoblotted with specific antibodies. The anti-phospho-FGFR1 and anti-FGFR1 antibodies were purchased from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Paso Robles, CA), respectively. All other antibodies were obtained from Cell Signaling (Danvers, MA).

Western blot analysis was followed by the standard protocol as we previously described [47 (link)]. Protein samples were extracted from the stored hippocampus tissues, and they were homogenized in lysis buffer (Cell Signaling Technology) with protease inhibitor cocktail (Life Technologies) and phosphatase inhibitor cocktail (Sigma). Proteins were separated by 10% SDS-PAGE eletrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4 °C with the primary antibodies: anti-FGF21 (1:1000, Abcam), anti-FGFR1 (1:200, Santa Cruz), anti-phospho- FGFR1 (1:1000, Abcam), anti-β-Klotho (1:1000, Abcam), anti-Akt (1:200, Santa Cruz), anti-phospho-Akt (1:100, Santa Cruz), anti-GSK-3β (1:1000, Cell Signaling), antiphospho- GSK-3β (1:1000, Cell Signaling), anti-IL-1β (1:1000, Abcam), anti-TNFα (1:1000, Abcam), anti-IL-6 (1:500, Novus), anti-PSD95 (1:1000, Abcam), antisynaptophysin (1:1000, Cell Signaling), and anti-actin (1:10,000, Sigma). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with enhanced chemiluminescence (GE Healthcare). Quantitative densitometry was performed on the protein bands by using ImageJ software.