Fl 218

HL-60 cells were treated with VitD for 72 hours and 30 × 10⁶ cells were collected and washed with ice cold PBS and resuspended in Buffer A (20 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl, 0.1% NP40, 0.2 mM EDTA, 10% glycerol). 800 μg of supernatant (cytoplasmic extract) were incubated with tRNA-saturated magnetic beads (Streptavidin MagneSphere Paramagnetic Particles, Promega) and ten biotinylated DNA probes in IP-Buffer (20 mM Hepes KOH pH 7.4, 150 mM KCl, 2 mM MgCl2, 0.01% Triton X-100, 5% glycerol, 1 mM DTT) for 1 hrs at R.T. Beads were washed four times with IP-Buffer and resuspended in 1 mL of Trizol and Protein loading dye for RNA and protein analysis, respectively. Protein levels were analyzed by western blot as previously described (Hughes et al., 2015). Antibodies utilized were against Ago2 (ab32381, Abcam) and HPRT (FL-218, Santacruz Biotechnology).

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).