Mouse fgf2

Experiments were performed on adult C57 black mice (4-8 weeks of age) according to the guidelines of the animal care and use committee of East Carolina University, an AAALAC-accredited facility. Mixed cortical/hippocampal neurons were isolated and cultured with minor modifications [31 (link)]. In brief, cells were released from dissected hippocampi and cortex by incubation with papain (Worthington Biochemical Corporation, Lakewood, NJ, USA) by gentle trituration. Subsequently, neuronal fractions from the cells were separated utilizing OptiPrep density gradient (Sigma, St. Louis, Mo, USA) and seeded in Neurobasal medium containing B27 supplement, 0.5 mM glutaMax, gentamycin, mouse FGF2, and PDGFbb (10 ng/mL) growth factors (Thermofisher Scientific, Waltham, MA, USA) onto 35 mm poly-D-lysine coated glass bottom dishes (MatTek, Ashland, MA,USA) and incubated at 37°C. Upon neurite and synaptic formation (7-10 DIV), cells were transfected with glycosylated (wt) and unglycosylated (N220/9Q) forms of Kv3.1b using Lipofectamine^® 2000 reagent (Thermofisher Scientific, Waltham, MA, USA) as described [18 (link)]. Cells greater than 48 h post transfections were utilized for live cell total internal reflection fluorescence (TIRF) microscopy studies, and to make whole cell lysates for western blots.