Sodium pyrophosphate

Human breast epithelial cells (MCF10A) were transfected with a pQCXIH vector and were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF, 0.5 μg/mL hydro-cortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, and 50 μg/mL penicillin/streptomycin until 70–80% confluence was reached. The cells were then lysed in a buffer containing 8 M urea, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 50 mM ammonium bicarbonate, one-third tablet of protease inhibitor, and 2 mM sodium ortho-vanadate. Proteins were denatured, reduced, and alkylated after which tryptic digestions were performed at an enzyme/substrate ratio of 1:50. For method comparison between SCX and RP, digested peptides were cleaned by flowing through a 1 mL solid-phase extraction C18 column (Discovery DSC-18, SUPELCO, Bellefonte, PA). Samples were concentrated using a Speed-Vac SC 250 Express (Thermo Savant, Holbrook, NY) and stored at −80°C until time for analysis. A 300.0 μg desalted peptide sample was used for each SCX, low-pH RPLC, and high-pH RPLC fractionation. A 300.0 μg nondesalted protein digest was used to evaluate the potential of high-pH approach for desalting.

Our first immunoblotting study was aimed to investigate the influence of a history of cocaine self-administration experience upon the protein expression of mGluR1/5 within PFC subregions at early versus protracted withdrawal. For this study, within each cohort of rats, separate groups of Sal1h, Sal6h and Coc6h rats underwent a 2-h cue test session under extinction conditions at either 3 or 30 days withdrawal (n=12 per group at the start of the experiment) to assay for cue-reinforced lever-pressing behavior. Immediately upon completion of this cue test, animals were killed by rapid decapitation. The dmPFC (anterior cingulate and dorsal prelimbic cortices) and vmPFC (ventral prelimbic and infralimbic cortices) were dissected out over ice in a manner identical to that described by others (Ghasemzadeh et al., 2009 (link)). The tissue derived from animals in this study was immunoblotted in 2 separate ways. First, comparisons were made across the tissue from the 3 different self-administration groups separately for each time-point (i.e., Sal1h, Sal6h & Coc6h rats sacrificed at 3 days withdrawal were compared in one assay, and a parallel assay compared Sal1h, Sal6h & Coc6h rats sacrificed at 30 days withdrawal in a separate assay). This “single time-point” analysis was done to enable a comparison of protein expression between the SAL1h and SAL6h rats at each time-point, as well as to determine whether or not a history of cocaine self-administration influenced protein expression relative to a history of saline self-administration at that particular withdrawal time-point. For these assays, the immunoreactivity of the mGluR1 and mGluR5 bands from the Sal6h and Coc6h animals was normalized to the average of that for the Sal1h animals run on the corresponding gel (n=4–5/gel) and the data expressed as a percentage of that averaged Sal1h signal for each gel. The data for the 3-day and for the 30-day assays were analyzed separately using an univariate analysis of variance (ANOVA) across the 3 different self-administration groups to determine the presence/absence of a cocaine effect.
The next analysis examined for proteomic correlates of the increased lever-pressing exhibited by both saline and cocaine self-administering animals at 30 days of withdrawal, by comparing tissue from the Sal6h and Coc6h animals sacrificed at both withdrawal time-points on the same gel. For this “time-course” assay, the immunoreactivity of the mGluR1 and mGluR5 bands were normalized to the average of the Sal6h-3 day withdrawal group run on the corresponding gel (n=3–4/gel) and the data expressed as a percentage of that averaged signal for each gel. The data were analyzed using orthogonal comparisons, within each self-administration group, to determine the presence/absence of time-dependent changes in protein expression. A second, follow-up, study assayed vmPFC and dmPFC tissue from Sal1h and Coc6h rats, sacrificed at 3 versus 30 days withdrawal, in the absence of any cue testing. This study was conducted to determine the extent to which our cocaine-induced changes in mGluR1/5 protein expression reflected alterations in the basal expression pattern of these receptors or some interaction between cocaine experience and the cue-reinforced testing conditions of our animals. The data analysis for this study was identical to that employed for the “time-course” study above.
The immunblotting procedures for detection of total mGluR1 and mGluR5 levels in brain tissue homogenate were identical to those described recently by our group (e.g., Cozzoli et al., 2009 (link), 2012 ; Goulding et al., 2011 (link)). In brief, tissue was homogenized in a solution consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 1 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, 1 mM orthovanadate, and 2 μM microcystin LR were included to inhibit phosphotases. After centrifugation at 10,000 g for 20 min, the supernatant of the homogenates were quantified for protein content using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) and stored at −80 °C. Protein samples (20 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis on Tris-Acetate gradient gels (3–8%) (Invitrogen, Carlsbad, CA). Wet polyvinylidene difluoride (PVDF) (Bio-Rad) membrane transfer was employed, and membranes were pre-blocked with either phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dried milk powder for a minimum of 2 hrs before overnight incubation with primary antibody. An anti-mGluR5 rabbit polyclonal antibody (Millipore, Billerica, MA; 1:1000 dilution) and an anti-mGluR1a mouse polyclonal antibody (Millipore; 1:500 dilution) were used for receptor detection. A rabbit primary anti-calnexin antibody (Stressgen, Ann Arbor, MI; 1:1000 dilution) was used as a control to ensure even protein loading and transfer. Membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Millipore; 1:5,000–1:10,000 dilution) or anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA; 1:10,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). Image J (NIH, Bethesda, MD) was used to quantify immunoreactivity levels.

Western blot analysis was carried out as previously described (Maguire and Mody, 2007 (link);Maguire et al., 2009 (link);Maguire et al., 2005 (link)). Animals were anesthetized with isoflurane, killed by decapitation, and the PVN, hippocampus, and cerebellum were rapidly removed. The tissue was sonicated in homogenization buffer (containing 10mM NaPO4, 100mM NaCl, 10mM sodium pyrophosphate, 25mM NaF, 5mM EDTA, 5mM EGTA, 2% Triton X-100, 0.5% Deoxycholate, 1mM sodium vanadate, pH 7.4) in the presence of protease inhibitors (complete mini, Roche, and fresh phenylmethylsulfonyl fluoride (PMSF)). The lysate was incubated on ice for 30 min then the supernatant was collected following centrifugation at 14,000 rpm for 10 min at 4°C. Protein concentrations were determined using the DC Protein Assay (BioRad). Total protein (100 μg for the GABA_AR δ subunit and 50 μg for KCC2 and Ser940) was loaded onto a 10% SDS–polyacrylamide gel, subjected to gel electrophoresis, transferred to a Immobilon-P membrane (Millipore), blocked in 10% non–fat milk, and probed with a monoclonal antibody specific for the GABA_AR δ subunit (1:500, PhosphoSolutions 868-GDN), KCC2 (1:1000, Millipore), or Ser940 (1:1000, a generous gift from Dr. Steve Moss). The blots were incubated with peroxidase labeled anti–rabbit IgG (1:2000, GE Healthcare) and immunoreactive proteins were visualized using enhanced chemiluminescence (Amersham). Optical density measurements were determined using the NIH Image J software.
For biotinylation experiments, slices containing the PVN were incubated in 1mg/ml NHS-biotin (Pierce) in normal artificial cerebral spinal fluid (nACSF) for 30 min on ice. The slices were then washed thoroughly with ice cold nACSF and the total protein was isolated and quantified as described above. 100μg of total protein was incubated with 50μl of streptavidin magnetic beads (Pierce) in 1ml phosphate-buffered saline (PBS) overnight at 4°C. The solution was centrifuged and the pellet was thoroughly washed and resuspended in 50μl loading buffer. The proteins were eluted from the magnetic beads which were removed by centrifugation, and 20μl of the loading buffer/protein solution was loaded onto a polyacrylamide gel and proteins visualized as described above.