Irdye 680 goat anti mouse igg

Total protein lysates for immunoblotting were obtained using SDS lysisbuffer (10 mM Tris-HCl [pH 7.5], 1% SDS, and 2 mM EDTA) supplemented with protease inhibitors (1 mM PMSF, 50 mM NaF, 50 µg/ml leupeptin, 1 mM orthovanadate, 4 µg/ml aprotinin). Protein concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific #23227F). 30 µg of protein were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Antibodies used were phospho-ERK1/2 (Thr202/Y204; Cell Signaling #4370), Ras (Thermo Scientific #1862335) and GAPDH (Ambion Am4300). Secondary antibodies were IRDye 800CW goat anti-rabbit IgG (LI-COR Bioscience 926-32211), IRDye 800CW goat anti-mouse IgG (LI-COR Bioscience 92632210) and IRDye 680 goat anti-mouse IgG (LI-COR Bioscience 926-322220). Blots were developed using the LI-COR Odyssey system.

Choroid plexus samples were lysed in lysis buffer (63 mM Tris–HCI, 2% SDS, 0.1% 2‐mercaptoethanol), and protein concentration was determined using the BCA kit (23225ZZ, Life Technologies). Forty micrograms of choroid plexus lysate and EVs isolated from 25 μl of CSF were loaded onto a 15% SDS–PAGE gel. Proteins were separated on the gel at 100 V for 3 h. Proteins were transferred to 0.2 μm nitrocellulose membranes (NBA083G, Perkin Elmer), and membranes were blocked using Odyssey blocking buffer (927‐40000, Li‐Cor). Next, membranes were incubated with TTR (1:1,000; A002, DAKO) and β‐actin (1:5,000; 691002, MP Biomedicals) antibodies diluted in Odyssey blocking buffer. After washing, blots were incubated with IRDye 800CW goat anti‐rabbit IgG (1:10,000; LI 926‐32211, Li‐Cor) or IRDye 680 goat anti‐mouse IgG (1:10,000; LI 926‐68070, Li‐Cor) and imaging was done using the Odyssey imaging system.

Total protein was isolated from cortical tissue of EGFP^+/+, EGFP^–/+, and EGFP^−/− (wild-type) rats at P18 by homogenizing in 10 mm Tris-HCl, pH 7.6/150 mm NaCl/1% Nonidet P-40/1% sodium deoxycholate, with protease and phosphatase inhibitors (Sigma-Aldrich). For each sample, 30 μg of cortical protein homogenate was resolved on a 12% Bis-Tris polyacrylamide gel (NuPage, Thermo Fisher Scientific) under reducing and denaturing conditions, and transferred to PVDF membrane. Membranes were blocked with Odyssey Blocking Buffer (LI-COR) diluted 1:1 with Tris-buffered saline (TBS), pH 7.4, and incubated overnight at 4°C with primary antibodies diluted in a 1:1 solution of Blocking Buffer/TBS-0.01% Tween. The primary antibodies used were chicken-anti-GFP (1:4000; Thermo Fisher Scientific) and mouse anti-Gapdh (1:5000; Abcam). After washing in TBS-0.1% Tween, membranes were incubated in 1:1 Blocking Buffer–TBS-Tween/0.02% SDS containing secondary antibodies (both diluted 1:10,000; IRDye 800 donkey-anti-chicken and IRDye 680 goat-anti-mouse IgG, LI-COR). Membranes were imaged on an Odyssey CLx Infrared Imaging System (LI-COR).

Human Type I PDZ GPCR cDNAs were purchased from the Missouri S&T cDNA resource center (cdna.org) or cloned from human brain cDNA library (provided by Professor Ning Zheng, HHMI, University of Washington Department of Pharmacology). Human α-syntrophin cDNA was purchased from OriGene Technologies, (Rockville, MD, USA). Human FLAG-Scribble was provided by Professor Jon Huibregtse (Department of Molecular Sciences, University of Texas at Austin). cDNAs were subcloned into pGlue (provided by Professor Randall T. Moon, HHMI, University of Washington Department of Pharmacology) using In-Fusion HD cloning technology (Clontech).
(R)-(−)-phenylephrine hydrochloride (P6126) was purchased from Sigma (St Louis, MO, USA). Anti-FLAG antibodies were purchased from Sigma (2368). Alexa fluor 633 goat-anti Rabbit IgG (A-21070) and Alexa fluor 568 Goat anti-rat (A-11011) antibodies from Life Technologies. Topro-3 iodide (T3605) is from Life Technologies. Rabbit polyclonal α-syntrophin (H-65, sc-50460), and rabbit polyclonal Scrib (H-300, sc-28737) antibodies from Santa Cruz Biotechnology. Mouse monoclonal Anti-syntrophin (1351, ab11425) antibody and rabbit polyclonal anti-Myc tag antibody (ab9106) from Abcam (Cambridge, MA, USA). Anti-HA mouse mAb (6E2, #2367) from Cell Signaling (Danvers, MA, USA). IRdye 680 goat antimouse IgG and IRdye 800cw goat antirabbit IgG from Li-Cor (Lincoln, NE, USA).