Nupage bis tris 4 12 protein gels

Whole lysate proteins were extracted from the olfactory bulb where GABAergic cells are highly enriched. The olfactory bulbs of Tsc1^Ctrl and Tg(Nkx2.1-Cre);Tsc1^flox/flox mice were dissected at P14 and P40 and snap frozen in liquid nitrogen. The tissue was then incubated in lysis buffer (150 mM sodium chloride, 1% Triton x-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM TrisHCl, pH 8, 2 mM EDTA supplemented with a protease inhibitor cocktail III (Calbiochem)). The concentration of total protein was measured using the Bradford assay (BioRad). Proteins were separated on Novex Tris-Glycine 16% or NuPage Bis-Tris 4–12% protein gels (Invitrogen) in SDS running buffer and were transferred to PVDF membranes (BioRad). The following primary antibodies were used: rabbit anti-LC3B (1:1000, Novus, Cat #NB100-2220), rabbit anti-p62 (1:500, Proteintech, Cat# 18420-1-AP), rabbit anti-pAMPK (1:800, T172, Cell Signaling, Cat# 2535), rabbit anti-AMPK (1:1000, Cell Signaling, Cat#2532), rabbit anti-ULK1 (1:1000, D8H5; Cell Signaling, Cat# 8054), rabbit anti-pULK1 (1:1000, Ser555, D1H4; Cell Signaling, Cat# 5869), and mouse anti-GAPDH (1:5000, ThermoFisher, Cat# MA5-15738). Bands were quantified using Image J software. The intensity of LC3 and p62 bands was normalized over the intensity of the GAPDH band. pAMPK and pULK1 bands were further normalized over total AMPK and ULK1 levels, respectively.