Proteinase inhibitor cocktail tablet

For Western blot analysis, H9c2 cells were washed with PBS and lysed in lysis buffer (50 mM Tris, pH 7.5; 0.5 M NaCl; 1.0 mM EDTA, pH 7.5; 10% glycerol; 1 mM basal medium Eagle; 1% Igepal-630; proteinase inhibitor cocktail tablet (Roche, Mannheim, Germany)) and the debris were pelleted by centrifuging at 12,000× g for 30 min. The supernatants were electrophoresed by sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto Immobilon™ PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked using 5% low-fat milk and 1% Tween 20 in PBS and the blotted proteins were probed with one of the following antibodies: anti-NFIL3, anti-Bax, anti-Bak, anti-Cyt C, anti-cleaved caspase-3, anti-α-tubulin, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IGF2, anti-IGF2R (Abcam, Cambridge, MA, USA), anti-IGF1R, anti-phospho-IGF1R, anti-PI3K, anti-phospho-Akt, and anti-phospho-PLCβ (Cell Signaling Technology, Beverly, MA, USA). Protein expression was detected with the ECL detection system (Millipore).

Protein was extracted from frozen kidney tissues using RIPA lysis buffer (P0013B; Beyotime, Nantong, China) with a phosphatase inhibitor tablet (Roche Diagnostics GmbH, Mannheim, Germany), a proteinase inhibitor cocktail tablet (Roche), and 100 mM phenylmethanesulfonyl fluoride (ST506; Beyotime), and the ratio of the listed chemicals to the RIPA lysis buffer was 1:100. Protein concentration was determined using the Bradford protein assay (P0011; Beyotime). After the proteins were separated via SDS-PAGE, they were transferred electrophoretically to PVDF membranes (Millipore, Billerica, MA) and blocked with 5% nonfat milk or BSA. Then, immunoblotting was performed using specific antibodies overnight at 4 °C: rabbit anti-PGIS (1:500; Abcam), anti-PKA (1:1000; CST, Danvers, MA), anti-P-PKA (1:1000; CST), anti-β-actin (1:5000; CST), and anti-α-tubulin (1:8000; Abcam). Then, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig for 1 h at room temperature. Antibody labeling on the Western blots was visualized using a chemiluminescence reagent (WBKLS0100; Millipore) with GE ImageQuant LAS 4000. Densitometry analysis was performed using ImageJ software.

Human saliva was obtained from Lee Biosolutions. Saliva (10 mL) was diluted 1:2 in PBS and centrifuged at 5,500 rpm for 15 min at 4 °C. The supernatant was collected and a proteinase inhibitor cocktail tablet (Roche #11836170001) was added, then the solution was filter-sterilized through a 0.22 µM filter (Millex). The conditioned saliva was used to coat wells of 24-well tissue culture-treated plates (Falcon) at 37°C for 30 min and residual saliva was washed off the wells using sterile PBS.

THP-1 cells (1 × 10⁷/well) were lysed with lysis buffer [50 mM Tris HCl, pH 8.0; 137 mM NaCl; 1 mM EDTA; 1% (vol/vol) Triton X-100; 10% (vol/vol) glycerol; 1 mM PMSF; 1 μg/mL each of aprotinin, leupeptin, and pepstatin; 1 mM Na₃VO₄; 1 mM NaF; and proteinase inhibitor cocktail tablet (Roche, Basel, Switzerland)] for 20 min on ice. The lysate was cleared of cell debris by centrifugation at 17,000 xg for 10 min at 4 °C. The cell lysate and 20 μg His-tagged Rv3463 protein were mixed and incubated at 4 °C for 6 h, and then His-tagged Rv3463 (His)-, TLR2- and TLR4-associated proteins were immunoprecipitated by incubation with Ni-NTA Agarose (Qiagen, Hilden, Germany) or Dynabeads®Protein A (Thermo Fisher Scientific) for 24 h at 4 °C after incubation with an anti-mouse IgG Ab as a control Ab for anti-Rv3463 (His), anti-rabbit IgG Ab as a control Ab for the anti-TLR2 and anti-TLR4 for 4 h at 4 °C. The beads were collected, washed and boiled in 5x sample buffer for 5 min. The bound proteins were analyzed on by immunoblotting with anti-TLR2, anti-TLR4, and anti-His Abs.

To detect cytosolic cytochrome c, tissues were suspended in a buffer (50 mM Tris (pH 7.5), 0.5 M NaCl, 1.0 mM EDTA (pH 7.5), 10% glycerol and proteinase inhibitor cocktail tablet (Roche) for 3 min. on ice), homogenized by 40 strokes in a Dounce homogenizer, and centrifuged at 12,000× g for 15 min. The supernatant was the cytosol fraction, and the pellet was resuspended in lysis buffer as the membrane fraction.