Western lightning plus ecl

Protein (100 μg) was mixed at a 1:1 v/v ratio with 2× sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 6 M urea; 10% β-mercaptoethanol; and 20% glycerol) and heated to 100 °C for 5 min. Samples were electrophoresed on 12% Tris-glycine SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (GE Healthcare, Marlborough, MA, USA). Membranes were blocked with 5% skim milk (Thermo Fisher Scientific) or 3% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) in PBS (pH 7.4) containing 0.05% TWEEN^® 20 (PBST) for 3 h at RT. Primary antibodies (1:1000; against STAT1/3, JAK2, SOCS1, TYK2, TAK1, SHP-2, CSF-1R, NF-κB1, MyD88, ERK1/2, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were prepared in either 2% skim milk or 0.5% BSA in PBST overnight at 4 °C. Membranes were washed with PBST and treated with the HRP-linked anti-rabbit secondary antibodies (1:4000; Sigma-Aldrich) or goat anti-mouse IgG HRP conjugate (1:4000; Thermo Fisher Scientific) in 2% skim milk or 0.5% BSA in PBST for 2 h at RT. Subsequently, the membranes were developed using western Lightning^® Plus-ECL (Thermo Fisher Scientific) on Hyperfilm™ (GE Healthcare).

Equipment and reagents used for Western blots were from Bio-Rad, Mississauga, ON, Canada unless otherwise stated. Thirteen µg of purified EV total protein (lysed in RIPA or NP40 buffer), and 4X Laemmli sample buffer loading dye with or without 10% β-mercaptoethanol, respectively, were electrophoresed in 1x TG-SDS Buffer on TGX Stain-Free™ Precast gels. Following electrophoresis, UV activation of the gel was performed and the gels were imaged using the manufacturer’s protocol. Proteins were transferred onto low-fluorescence PVDF membrane using the Trans-Blot Turbo Transfer System. Total protein intensity was imaged using ChemiDoc™ MP system according to manufacturer’s protocol. Membranes were blocked in 5% milk/TBS-T for 1 hour at room temperature. All primary antibodies were incubated overnight at 4 °C in 5% milk/TBS-T. Following washes with TBS-T, membranes were incubated for 1 hour at RT in the appropriate secondary antibodies diluted to 1:3,000 in 5% milk-TBST. Membranes were developed using Western Lightning Plus-ECL (Thermo Scientific) using the ChemiDoc™ MP system. Band intensities within the linear range were quantified using Image Lab software. Proteins of interest were normalized to total protein intensity in place of housekeeping genes.

Western blotting was performed as described previously (Brown et al., 2009 (link)). Briefly, cells isolated from or 5 mg/ml Collagen (obtained from Dr. Jason Spector) were lysed in RIPA lysis buffer (Sigma-Aldrich) supplemented with 100x Protease/Phosphatase inhibitor cocktail (Cell Signaling Technology Inc). Cell extracts (20 μg per lane) were separated by NuPAGE 4–12% Bis-Tris protein gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes. The following primary antibodies were used: CDK4, Cyclin D3, phospho-Rb (Ser795), Rb, Bcl-2, Bax, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), phospho-p90RSK (Ser380), RSK1/RSK2/RSK3, c-Myc, phospho-Akt (Ser473), Akt, phospho-p70 S6 Kinase (Thr389), p70 S6 Kinase. Bound antibodies were revealed with HRP conjugated secondary antibodies. The following secondary antibodies were used: Goat anti-mouse IgG H and L (HRP), Donkey anti- Rabbit IgG H and L (HRP). Detected bands were consistent with expected molecular weights based on antibody datasheets (Key Resource Table). β–actin was used as a loading control. See Key Resources Table for antibody details used in this study. Membranes were scanned using the western lightning plus-ECL (Thermo Fisher Scientific). Signal intensities were quantified using ImageLab software (BioRad).

Mouse hippocampal tissues were lysed with a lysis buffer (Pierce IP Lysis Buffer, 87788, Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor cocktail (78442, Thermo Fisher Scientific). The tissue lysates were homogenized with a Tissue Grinder (10 strokes, 02-911-529, Thermo Fisher Scientific) and centrifuged at 800 × g for 5 min. Protein levels in the supernatant were measured by the BCA method. The samples were mixed with the standard protein sample buffer and boiled on a hot plate for 2 min, and subjected to SDS-PAGE with 4–20% Novex Tris-Glycine gels (Sigma-Aldrich), followed by protein transfer onto a nitrocellulose membrane. The membranes were Immunoblotted with incubation of anti-Ahnak (rabbit polyclonal, RU2024, 1:5,000) or Anti-Gapdh (mouse monoclonal, MAB374, Millipore, 1:5,000) primary antibody followed by incubation of horseradish peroxidase-linked goat anti-rabbit or anti-mouse secondary antibody (1: 5,000, Thermo Fisher Scientific). Antibody binding was detected using the enhanced chemiluminescence immunoblotting detection system (Perkin Elmer LLC WESTERN LIGHTNING PLUS-ECL, Thermo Fisher Scientific) and Kodak autoradiography film. The bands were quantified by densitometry using NIH Image 1.63 software.