Mammalian protease inhibitor

HEK293T cells were transfected with a total of 2.4 µg of DNA per 1×10⁶ cells. At 24 h after transfection, the cells were washed in cold PBS and lysed in RIPA lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, 1% sodium deoxycholate) or in NP40-lysis buffer (0.5% NP40, 25 mM Tris-HCl pH7.5, 100 mM NaCl, 50 mM NaF) supplemented with 10 mM N-ethylmaleimide, mammalian protease inhibitor (Sigma) and phosphatase inhibitor cocktail (Roche). Lysates were incubated with primary antibodies and protein-G– or protein-A–agarose beads (P4691 and P2670, Sigma) or with anti-FLAG M2 Affinity Gel (Sigma, A2220) for 1 h at 4°C. Beads were washed with RIPA or YP-IP buffer (0.1% NP40, 25 mM Tris-HCl pH 7.5 and 150 mM NaCl) 3–5 times and once with 10 mM Tris-HCl pH 7.5. Immunoprecipitated samples and 10 µg of the input were evaluated by western blotting.

Sf9 insect cells at 1.0 x 10⁶ cells/mL were co-infected with Multibac baculoviruses expressing FLAG-BRCA1^{Δexon 11} and StrepII-BARD1 for 48 hrs. Cells were harvested by centrifugation at 500 x g for 5 min at 4 °C. The cell pellet was resuspended in buffer E (50 mM TEA pH 7.4, 150 mM NaCl, 10% glycerol) supplemented with 1 mM EDTA and 1X mammalian protease inhibitor (Sigma-Aldrich) prior to lysis by sonication. The lysate was clarified by centrifugation at 16,000 x g at 4 °C for 30 min. The clarified lysate was gently mixed with Streptactin resin (GE Healthcare) pre-equilibrated in buffer E for 2 hrs at 4 °C. The loaded resin was washed in batch with 20 CV buffer E five times for 5 min each wash, and the protein was eluted in buffer E containing 0.5 mg/mL desthiobiotin (Sigma-Aldrich). The purified proteins were aliquoted and plunge-cooled in liquid nitrogen prior to storage at -80 °C.

Cytoplasmic and mitochondrial enrichments were collected using the mitochondrial isolation kit for tissue (Abcam, Cambridge, UK). RV specimens were homogenized with a Dounce homogenizer in isolation buffer supplemented with mammalian protease inhibitor (Sigma-Aldrich) and 100 µM PUGNAc (Sigma-Aldrich). After completing the centrifugation steps, the supernatant (cytoplasm) was saved for analysis. The final mitochondrial pellet was resuspended in SDS buffer [31 (link)] supplemented with protease inhibitor (Sigma-Aldrich). Protein concentration was determined using a BCA kit and equivalent amounts of protein were subjected to Western blot analysis as described above.

After KA seizure induction, animals were sacrificed, whole hippocampi hemispheres were harvested, and flash frozen in liquid nitrogen. A single hippocampal hemisphere was lysed in Radioimmunoprecipitation Assay buffer (RIPA: 50mM Tris, 150mM NaCl, 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with mammalian protease inhibitor (1:100, Sigma) and phosphatase inhibitors (10mM NaF, 2mM Na Orthovanadate, 4mM Na pyrophosphate, 10mM Na β-glycerophosphate). Tissue was homogenized using a probe sonicator (Fisher Scientific, Sonic Dismembrator, Model 100, Hampton, NH) by sonicating on power 4 for three rounds with 10 pulses each round. Samples were then centrifuged at 13.5 x g for 30 minutes to separate cell debris. Supernatants were isolated and quantified using the DC Protein Assay (Bio-Rad, Hercules, CA). 5X loading buffer (0.5M Tris, 10% SDS, 50% glycerol, 10mM EDTA and 1% B-mercaptoethanol) was added to each sample to reach a 1X final concentration. Protein extracts were boiled at 95˚C for 5 minutes and stored at -80˚C until run on an SDS PAGE gel.

The colon tissue samples were lysed in a RIPA buffer containing a 2% phosphatase-inhibitor (Thermo Fisher Scientific, Waltham, MA, United States) and a 1% mammalian-protease inhibitor (Sigma-Aldrich). 50 μg of extracted proteins were run a single track of 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred electrophoretically onto cellulose membranes. The membranes were blocked in 5% non-fat dry milk for 60 min, and then incubated with primary antibodies against nNOS (1:1000; Cell Signaling, Boston, MA, United States), Sub-P (1:100; Novus biologicals, Centennial, CO, United States), ChAT (1:100), NGF (1:200), COX2 (1:300), Tryptase (1:500), PGP9.5 (1:1000) and GAPDH (1:10,000) (all Abcam, Cambridge, United Kingdom), and PGE2 polyclonal antibody (1 : 200; Bioss Inc., Woburn, MA, United States) (Chia et al., 2017 (link)) overnight at 4°C. The membranes were washed 3× with TBS-T (TBS mixed with 0.1% Tween-20) and then, they were incubated with ECL AP-conjugated anti-rabbit/mouse IgG (1:3,000; GE Healthcare, United Kingdom) for 90 min in room temperature. Quantitative western blot results were obtained by densitometric analysis using image processing and analysis in Java (Image J, NIH, Bethesda, MD, United States). The percent change of relative intensity was calculated against control samples.