Protease phosphatase inhibitor cocktail

We performed coimmunoprecipitation with rat brains as described previously [22 (link)]. After rats were anesthetized and decapitated, brains were removed. The cerebellum was homogenized in cold isotonic sucrose homogenization buffer containing 0.32 M sucrose, 10 mM HEPES, pH 7.4, 2 mM ethylenediaminetetraacetic acid (EDTA), and a protease/phosphatase inhibitor cocktail (Thermo Scientific, Rochester, NY). Homogenates were centrifuged at 800×g for 10 min at 4 °C. P2 pellets (synaptosomal fraction) were obtained by centrifuging the supernatant for 15 min (10,000×g) at 4 °C. Washed P2 was resuspended in the sucrose homogenization buffer containing Triton X-100 (0.5 %, v/v). The suspension was lysed and incubated with gentle rotation for 20 min at 4 °C. Samples were centrifuged for 20 min at 32,000×g to yield the pellet enriched with synaptic membranes. These pellets were solubilized in sucrose-Triton buffer containing 1 % sodium deoxycholate and a protease/phosphatase inhibitor cocktail for 1 h at 4 °C. Solubilized proteins were incubated with a rabbit antibody against ERK1/2 or mGluR1a. The complex was precipitated with 50 % protein A or G agarose/sepharose bead slurry (GE Healthcare). Proteins were separated on Novex 4–12 % gels and probed with a mouse antibody against ERK1/2 or mGluR1a.

Sub-confluent cells were treated with 0, 2.5 and 5μM of OSU-T315 for 48 and 72 hours for cell lines, and 72 hours for primary cells. Cells were then harvested and homogenized in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris, pH 8.1) supplemented with protease/phosphatase inhibitor cocktail (Thermo Scientific, Waltham MA). Electrophoresis was performed with 25μg or 50μg of protein/lane in a 4-20% gradient SDS- polyacrylamide gel (Thermo Scientific, Waltham MA). Proteins were transferred to PVDF membranes (Millipore, Billerica MA), and probed with specific antibodies of interest and horseradish peroxidase-conjugated secondary antibodies. The chemiluminescent signals were detected in X-ray films.

LC and DR tissue punches were homogenized in 200 μL of tissue lysis buffer (500 mM NaCl, 50 mM Tris HCl pH 7.6, 10% NP-40, 70% glycerol) with 0.02% protease phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA) in conjunction with mechanical disruption with a bullet blender (Next Advance, Averill Park, NY) at 4°C. Protein concentrations of the supernatant were assessed using a BCA assay (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol and read using a Synergy 2 Multi-Mode plate reader (Bio Tek, Winooski, VT) with Gen5 software (Bio Tek, Winooski, VT). The remaining tissue homogenate was stored at −80°C.

Cells were harvested and lysed in RIPA buffer (50mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate) in the presence of 1X protease/phosphatase inhibitor cocktail (Thermo Fisher) on ice for 15 min. Patient samples and xenografted tumors were homogenized in liquid nitrogen and protein was extracted by T-PER protein extraction buffer. Chromatin and cell debris were then pelleted down by centrifuging at 12,000 rpm at 4°C. The supernatant was collected, and BCA assay was used to quantify protein concentration. Protein lysates were fully denatured by 1X Laemmli sample buffer and boiled at 95°C for 10min. Protein lysates were then separated in SDS-PAGE gel and blotted onto PVDF membrane. Membrane was then blocked by 5% non-fat milk in TBS/T (20mM Tris pH 7.4, 150 mM NaCl, 0.1% (w/v) Tween-20) for at least 1hr before incubation with primary antibody at 4°C for overnight. The blot was then probed with HRP conjugated secondary antibody and developed using ECL reagent. Subcellular fractionation was performed according to manufacturer’s instruction. The protein concentration of each fraction was quantified by BCA assay.