Phosphatase inhibitor tablet

The protein expressions of p65, p-p65, IκBα, Nrf2, and HO-1 were determined by Western blot analysis. Antibodies against p65, p-p65 and IκBα were purchased from Cell Signaling (Beverly, MA, USA), antibody against Nrf2 was purchased from Ruiying Biological, and antibodies against GAPDH, β-actin and β-tubulin were purchased from Bioworld. Tissues were lysed in RIPA buffer (Applygen Technologies) with phosphatase inhibitor tablet (Roche Diagnostics) and protease inhibitor (Cocktails, AMRESCO). Lysates were then centrifuged for 15 min at 12,000 × g at 4 °C and supernatants were collected and protein concentrations were determined by BCA Protein Assay Reagent (Pierce). Cell lysates (15-30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to nitrocellulose membranes (Millipore). For western blotting analysis, membranes were incubated with primary antibodies for overnight at 4ºC followed by incubation with a secondary antibody for 1h at r.t. Then the signals were detected by enhanced chemiluminescence according to the manufacturer’s recommendation.

To prepare human cell lysate, approximately 1 × 10⁷ HEPG2 or 3T3 cells were collected by centrifugation at 4 °C, washed in cold PBS and resuspended in 250 μL binding buffer (20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.01% Triton X-100) supplemented fresh with a protease inhibitor cocktail tablet (Roche; 1183617001), phosphatase inhibitor tablet (Roche; 04906837001), 20 mM Na₂MoO₄, and 1 mM DTT. After vortexing, cell lysate was frozen at −80 °C for 2 h, followed by thawing on ice. The vortex-freeze-thaw step was repeated. Complete lysis was confirmed by microscopy followed by centrifugation at 14,000 rpm for 30 min at 4 °C. To prepare yeast lysates, an overnight culture grown in YPD was diluted to an OD₆₀₀ of 0.15 in 1 L of pre-warmed YPD. This culture was grown for 4 h at 37 °C, then centrifuged at 4000 rpm for 20 min at 4 °C. The pellet was resuspended in 10 mL of the same binding buffer used to prepare mammalian lysate, passed twice through a French Press and then centrifuged at 14,000 rpm for 30 min at 4 °C. Post centrifugation, yeast and mammalian lysates were filtered (0.22 μm), supplemented with sterile glycerol to a final concentration of 15% (v/v), flash frozen, and stored at −80 °C for up to 5 months. Aliquots were thawed immediately prior to use.

Cells were washed twice with ice-cold 1× PBS, harvested, and lysed in RIPA buffer (Applygen Technologies) with phosphatase inhibitor tablet (Roche Diagnostics) and protease inhibitor (Cocktails, AMRESCO). Cell lysates were then centrifuged for 15 min at 13,000 × g at 4 °C and supernatants were collected and protein concentrations were determined by BCA Protein Assay Reagent (Pierce). Cell lysates (15–30 μg) were subjected to 8–12% SDS-polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to nitrocellulose membranes (Millipore). For western blotting analysis, membranes were incubated with primary antibodies for overnight at 4°C followed by incubation with a secondary antibody for 1h at r.t. Then the signals were detected by enhanced chemiluminescence or fluorescence according to the manufacturer’s recommendation.

To extract cellular protein, cardiac fibroblasts (2 hours after Ang II stimulation) were lysed with “M-PER Mammalian Protein Extraction Reagent” (Thermo Scientific) containing an EDTA-free protease inhibitor cocktail tablet (Roche Diagnostic, Basel, Switzerland) and a phosphatase inhibitor tablet (Roche Diagnostic, Basel, Switzerland). For extraction of cardiac protein, frozen peri-infarct zone tissues from apex-level heart slices harvested from the MI + KLH group on day 56 were homogenized in lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS)) containing a protease inhibitor cocktail tablet. The lysates were centrifuged at 14000 × g for 15 minutes. The protein concentrations of all samples including culture supernatant were measured using the “Bio-Rad Protein Assay Kits” (Bio-Rad, Milan, Italy) to equalize the protein concentrations. These protein samples were stored at −80 °C, and were used as samples for Western blotting and gelatin zymography.

Brain Aβ_1–40 and Aβ_1–42 species were detected by a three-step extraction protocol with modifications (65 (link)). Brains were homogenized using TissueLyser LT (Qiagen) in Tris-buffered saline (TBS: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing protease inhibitor mixture (Sigma-Aldrich) and phosphatase inhibitor tablet (Roche), centrifuged at 18,800g for 60 min at 4 °C, and supernatants were collected (representing the TBS-soluble fraction). The resulting pellets were treated with TNE buffer (10 mM Tris-HCl, 1% Nonidet P-40, 1 mM EDTA, and 150 mM NaCl) with protease and phosphatase inhibitors, and homogenized using TissueLyser LT. Homogenates were then centrifuged at 18,800g for 30 min at 4 °C, and supernatants were collected (representing the detergent-soluble fraction). The remaining pellets were treated with 5 M guanidine-HCl and dissolved by occasional mixing on ice for 30 min and centrifuged at 18,800g for 30 min at 4 °C. Supernatants were then collected; this is taken as the guanidine-HCl-soluble fraction. Aβ_1–40 and Aβ_1–42 species were separately quantified in each sample in duplicate by sandwich ELISA (IBL) (66 (link)). Aβ oligomers were quantified in the TBS-soluble fraction in duplicate by human Aβ oligomers (82E1-specific) assay (IBL) (67 (link)). All samples fell within the linear range of the standard curve.