Coomassie plus assay

PBS-perfused rat brains were dissected into cortex, hippocampus, and cerebellum. Tissue lysates from these regions were extracted in radioimmunoprecipitation assay (RIPA, 1X) lysis buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitors (Aprotinin 2 μg/ml, Leupeptin 5 μg/ml, Pepstatin 1 μg/ml, and PMSF 1 mM). Protein concentration was measured using Coomassie Plus Assay (Bradford method) reagent according to the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL). 50 μg proteins were then separated using 10% SDS-PAGE gels and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. Equivalent sample loading and transfer was confirmed by Ponceau S staining. Membranes were then probed overnight with human serum (1:5000) followed by incubation with HRP-conjugated secondary goat anti-human (1:5000, 401445, polyclonal Calbiochem, Darmstadt, Germany) antibody. Protein bands were visualized using Pierce enhanced chemiluminescence (ECL) Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL). Kodak X-ray film developers were used to develop the X-ray films in the dark room. The densitometry analysis of bands was performed using ImageJ software following the standard protocol provided by National Institutes of Health (NIH).

Protein concentrations were determined by Coomassie-plus assay (Thermo Scientific), and samples were diluted with buffer CSBL1 to 0.15 mg/mL. The 60 validated signaling pathway markers listed in Table S1 were profiled in a standard ZeptoMARK, as previously described.⁶⁵ (link) Briefly, analysis of raw excitation light intensity data was conducted by normalizing the net signal intensity of each sample. For each lysate sample represented by a total of four spots at four dilutions, a mean referenced fluorescence intensity (RFI value) was calculated based on a weighted linear fit through the four normalized sample spots and markers, with negative RFI values excluded. These RFI values were subsequently normalized against Prohibitin housekeeping protein prior to the comparison of analytes across the entire sample series. For heatmap analysis, each treatment group was compared to the normalized RFI values of RISC-free siRNA control group.

cIEC pellets isolated from 4 to 5 mice per group (time point) per biological replicate were individually solubilized using probe sonication in lysis buffer (100 mM triethylammonium bicarbonate [TEAB], 1% sodium deoxycholate [SDC], 10% isopropanol, 50 mM NaCl) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific), boiled for 5 min at 90°C, and sonicated once more. The protein concentration was determined with a Coomassie Plus assay (Thermo Scientific) according to the manufacturer’s protocol. Equal amounts of protein from each mouse from each group were combined. A total of 100 μg of protein per group was reduced with 5 mM Tris-2-carboxyethyl phosphine (TCEP) for 1 h, followed by alkylation with 10 mM iodoacetamide (IAA) for 30 min, and then digested by adding trypsin (Pierce) at a final concentration of 75 ng/μL to each sample and incubating the samples for 18 h at room temperature. Peptides were labeled with tandem mass tag (TMT) multiplex reagent (Thermo Scientific) for 1 h before quenching with a final volume of 5% hydroxylamine for 15 min (Sigma). TMT-labeled peptides were combined at equal amounts, and SDC was precipitated with formic acid (FA) at a final concentration of 2% (vol/vol) and centrifugation for 5 min at 10,000 rpm. The supernatant containing TMT-labeled peptides was dried with a centrifugal vacuum concentrator.

Animals received either A33 (0.3 mg/kg, 6 ml/kg, i.p.), or vehicle (5% DMSO in saline, 6 ml/kg, i.p.) at 30 min and 5 hrs post-surgery. At 6 hrs post-surgery the animals were anesthetized (3% isoflurane, 70% N₂O, 30% O₂, 5 min), decapitated and the ipsilateral parietal cortex was dissected at 4°C, snap frozen in liquid nitrogen and stored at -80°C. Samples were assayed in duplicate according to the manufacturer’s protocol using a cAMP ELISA (ADI-900-066, Enzo Life Science), and normalized to total protein using a Coomassie Plus assay (23236, ThermoFisher Scientific).