Epr5479

For determining the total expression of AMPA and phosphorylation of MAP2 and synapsin in LUHMES/RFP-derived neurons untreated or treated with TNF-α (10 pg/mL) or poly(I:C) (10 ng/mL) unexposed or not to C20 or HC69 cells untreated or treated with similar amount of TNF-α or poly (I:C), whole cell extracts (WCE) were prepared from 5 X 10⁵ neurons collected by gentle shaking and added to RIPA buffer (25 mM Tris, pH 7–8, 150 mM Na, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100). Protein concentration in WCE was measured by Bradford assay, and protein solutions were subjected to SDS-PAGE/Western blot using anti-glutamate receptor 1 (AMPA subtype) antibody [EPR5479] (Abcam, Cat. #ab109450), anti-MAP2 (phospho S136) antibody [EPR2361] (Abcam, Cat. #ab96378), and anti-synapsin I (Abcam, Cat. #phospho S9) antibody (Abcam, Cat. #ab194778). β-tubulin III (beta-TUJ) is a microtubule element of the tubulin family found exclusively in neurons [101 (link), 102 (link)], therefore, we used anti-beta-TUJ (Abcam, Cat. #ab18207) as loading control. These primary antibodies were bound by the appropriate IRDye 800CW secondary antibody, and the membranes were scanned and analyzed using the Odyssey Infrared Imaging System (LI-COR Biosciences, NE).