Bicinchoninic acid protein assay

Cells (1 × 10⁵ cells/mL) were cultured in 6-well culture plates and then treated with 0, 1, and 5 μg/mL 25-HC for 48 h. Thereafter, total proteins were extracted from the L929 cells using cell lysis buffer (Cell Signaling Technology), according to the manufacturer’s instructions. Protein concentration was determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific). Equal amounts of each protein sample were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA) at 4 °C. Thereafter, the PVDF membrane was blocked using 5% (v/v) bovine serum albumin (BSA; Sigma-Aldrich) prepared in Tris-buffered saline with Tween 20 (TBS-T; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then incubated with the primary antibodies diluted 1:1000 in TBS-T containing 5% (v/v) BSA at 4 °C for 12 h. The immunoreactive bands were visualized using the ECL System (Amersham Biosciences, Piscataway, NJ, USA) and exposed on radiographic film or MicorChemi 4.2 (Dong-Il Shimadzu Corp., Seoul, Korea).

For identifying CAQK binding proteins, mouse brains with brain injury were collected 6 h after injury. Using liquid nitrogen, the brains were crushed and ground into powder using a mortar and pestle. Next, brain tissue was lysed in PBS containing 200 mM n-octyl-beta-D-glucopyranoside and protease inhibitor cocktail (Roche) as described38 (link). The cleared lysate was loaded on to CAQK or control peptide (CGGK) coated Sulfolink-agarose beads (Pierce, Waltham, MA) and incubated at 4 °C for 3–4 h. The column was washed with wash buffer (75 mM octyl-beta-D-glucopyranoside and protease inhibitor cocktail in PBS) followed by washing with 0.5 mM control peptide in wash buffer to remove nonspecifically bound proteins. The bound proteins were eluted with 2 mM-free CAQK peptide. The eluted factions were pooled, their protein concentration determined by using bicinchoninic acid protein assay (Thermo Fischer) and the samples were digested using the filter-aided sample preparation method39 (link). Finally, the digested samples were desalted, dried and subjected to liquid chromatography–mass spectrometry (MS)/MS analysis at the Proteomics Core facility of the Sanford Burnham Prebys Medical Discovery Institute. All mass spectra were analysed with MaxQuant software version 1.5.0.25. The MS/MS spectra were searched against the Mus musculus Uniprot protein sequence database (version July 2014).

The tissue specimens were analyzed as previously described [10 ]. Briefly, the samples were homogenized in a glass homogenizer using 5.0 μL per mg of Radio-immunoprecipitation Assay Buffer (20–188 Millipore) that contained phosphatase and protease inhibitors (Thermo Scientific, Rockford, IL, USA). The homogenates were centrifuged at 4 °C (14,000 rpm for 10 min). Total protein content in the whole cell lysates was determined using the bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, USA). Twenty μg of the whole cell lysate was separated by electrophoresis, which was then transferred to PVDF (polyvinylidene difluoride) membrane. After blocking in 5% skimmed milk for one hour at 25 °C, the blot was incubated overnight at 4 °C with the mouse monoclonal anti-tyrosine hydroxylase (TH) antibody with a dilution of 1:1000 (ImmunoStar, WI, USA). After washing with PBS-T, the membrane was probed for one hour at 25 °C with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody with a dilution of 1:20,000 (Jackson ImmunoResearch, PA, USA). The bands of interest were visualized using a West Pico Chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). The blots were stripped and re-probed for GAPDH (1:1000; polyclonal Rabbit, Cell Signaling Technologies, USA) as a loading control [10 ]. Only samples positive for TH were used in this study.

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (65 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% v/v NP 40, 0.25% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid [EDTA], and 2 mM phenylmethylsulfonyl fluoride [PMSF]) for 30 min at 4°C. The lysate was clarified by centrifugation at 14,000 × g for 30 min at 4°C. The supernatant was collected, and the protein concentration was measured by the bicinchoninic acid protein assay (Thermo Fisher Scientific). The supernatant was diluted in Laemmli buffer that contained either 50 mM DTT or 50 mM IAA for reducing and non‐reducing conditions, respectively.