Nupage novex precast gels

Brain tissues were isolated from 8 weeks old Fgfr2^lox/lox (control), heterozyote Nestin-Cre;Fgfr2^+/lox and homozygote Nestin-Cre;Fgfr2^lox/lox mice, and homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Sodium deoxycholate, 1 mM EDTA, and Complete protease inhibitors (Roche)). Total protein concentration was determined with the Pierce BCA Protein Assay (Thermo Fisher Scientific), and 50 µg total protein per sample were separated in 10% NuPAGE Novex precast gels (Invitrogen) and blotted onto PVDF membranes (Hall/USA). Blots were blocked in 4% skim milk in TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) and probed with rabbit anti-Fgfr2 (1∶300; sc-122) and anti-hypoxanthine guanine phosphoribosyl transferase (Hprt) (1∶400; FL-218) antibodies (both from Santa Cruz Biotechnology). Membranes were developed in ECL substrate and exposed to Hyperfilm ECL (GE Healthcare).

Total protein extracts were assembled using RIPA lysis buffer Equal protein amounts (30-50 μg) were electrophoresed using NuPage Novex precast gels (Invitrogen/Life Technologies) and transferred with the iBlot dry blotting system (Invitrogen/Life Technologies). Bands were detected by ECL solution (Thermo Scientific, Waltham, MA, USA).