Cell lytic mt mammalian tissue extraction reagent

To confirm whether systemic dual-LPS administration induced signaling alterations in the mPFC and vHip, protein expression of inflammatory mediator NFκB was examined. This tissue preparation method differed from sample preparation for synaptosome enriched fractions (described below). In this non-behavior tested cohort, the mPFC (n = 4/treatment) and vHip (n = 3/treatment) of C57BL/6J mice treated with LPS (0.83 mg/kg) or vehicle were harvested at 24 h post T = 0 injection as described in Section 2.3 (Supplementary Fig. 1A). Each brain region was homogenized in a Storm 24 magnetic Bullet Blender for 4 min at a speed setting of 4 (Next Advance Inc., Averill Park NY, USA), with 0.5 mm zirconium oxide beads in combination with 50-70 μl of Cell-lytic MT mammalian tissue extraction reagent (Sigma-Aldrich) containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, and 0.1% SDS. The homogenization buffer contained Complete (Roche) protease inhibitor cocktail and phosphatase inhibitor cocktails Type II and III (Sigma-Aldrich). Homogenates were then centrifuged at 16,400 rpm (4°C) for 15 min and supernatants were collected for subsequent SDS PAGE and western blot analysis, as described below (Section 2.5.3).

Both ENT1^+/+ mice and ENT1^−/− mice were subjected to rapid CO₂ inhalation to induce unconsciousness, followed by decapitation and subsequent harvesting of brain for isolation of the NAc from both hemispheres under a surgical microscope (n = 4 per treatment in each genotype). The extracted tissue was snap-frozen on dry ice and stored at −80°C until it was processed for SDS-PAGE (Bio-Rad Criterion system). The NAc from each mouse was homogenized using Pellet Pestle (Fisher) in a Cell-lytic MT mammalian tissue extraction reagent (Sigma-Aldrich) containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, 0.1% SDS protease inhibitor cocktail type I (Roche) and II (Sigma). Whole NAc tissue lysates were then centrifuged at 500 g at 4°C and supernatants were collected. Protein concentration from each replicate supernatant was quantified using the Bradford protein assay (Bio-Rad). Tissue samples were loaded at 30 μg proteins/lane and separated in MOPS buffer via electrophoresis in a 4–12% Bis-Tris poly-acrylamide gel (Criterion, Bio-Rad, Hercules CA, USA) at 70 V for 20 min, followed by 140 V for 80 min.