P akt

The homogenized ventricles tissue or cultured cells were incubated in lysis buffer (pH 7.4) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 1% Triton-X 100, 1% NP-40, 0.1 mM Na₃VO₄, 12.5 mM b-glycerophosphate, 1 mM phenylmethanesulfonylfluoride (PMSF) and complete protease inhibitor cocktail. Insoluble heart tissues or cell fractions were removed by centrifugation at 13,000 × g, 4°C for 30 min. The concentration of the extracted proteins was measured by bicinchonininc acid assay (Sigma-Aldrich, St. Louis, MO, USA). The extracted proteins were boiled for 3–5 min. in 5× loading buffer. The expression of AT1 receptor, LC3b-II, beclin-1 and phosphorylation of Akt (p-Akt) were detected by immune-blotting with antibodies against AT1 receptor (catalogue no.sc-1173G; Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3b-II (catalogue no. 2775; Cell Signaling Technology, Danvers, MA, USA), beclin-1 (catalogue no. 3495; Cell Signaling Technology), and p-Akt (catalogue no. 9275; Cell Signaling Technology), and detection was performed by using an ECL Western-blotting Detection Reagents (RPN2106; GE Healthcare, Little Chalfont, Buckinghamshire, UK) visualized densitometry with LAS-300 Image software.

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibodies specific for fractalkine/CX3CL1, CX3CR1, ICAM-1, VCAM-1, p-PI3K p85α, PI3K p85α, p-Akt, Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human fractalkine/CX3CL1 was purchased from PeproTech (Rocky Hill, NJ, USA). The short hairpin RNA (shRNA) plasmid used for gene knockdown was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs used were ON-TARGETplus siRNAs and purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA).

Nuclear extracts, cytosol extracts, or total proteins of liver tissue were prepared as a published method with modification [19 (link)]. The protein concentration was measured by the BCA assay. Aliquots of protein (30 μg) were denatured at 95°C for 5 min before electrophoresis on 10% SDS-polyacrylamide gel. After transfer to a polyvinylidene difluoride (PVDF) membrane (Millipore), the blot was blocked with 5% nonfat milk solution for 1 h at room temperature and then incubated with a 1 : 1,000 dilution of primary antibodies selective against either NF-κB p65, total JNK, p-JNK, total ERK, p-ERK, total Akt, p-Akt, total p38, p-p38, histone, or β-actin (Santa Cruz Biotechnology) in Tris-buffered saline Tween-20 (TBST) at 4°C overnight, followed by 1 h at room temperature. The membrane was washed 3 times for 5 min each with TBST solution. The membranes were incubated with 1 : 10,000 dilution of horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection system and the band intensities were determined using a Gel Doc/ChemiDoc imager (Azure). The protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA).

Heart tissues (50–100 mg) were cut into small pieces and lysed with RIPA buffer (Roche, Switzerland), which consists of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail, at 4 °C, then ruptured by homogenizer on ice. The supernatant was collected after centrifugation at 14,000×g for 30 min at 4°C. The protein concentration of the cell lysate was quantified by using the bicinchoninic acid assay (Beyotime Biotechnology, Beijing, China).
Cardiac proteins from mice were subjected to polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes. The membranes were then probed with antibodies, including LC3-phosphatidyl ethanolamine conjugate (1:200), mTOR (1:1,000), GAPDH (1:2000) and PI3KIII (1:1,000) (all purchased from Cell signaling Technology, United States); BECN1 (1:500) and p-Akt (1:1,000) (all purchased from Santa Cruz); p62 (1:1,000), BAG3 (1:500) and HspB8 (1:500) (all purchased from Abcam, United Kingdom). Peroxidase activity was visualized with ECL (SantaCruz, United States). The bands were quantified using Quantity One.

In vivo ubiquitylation assays and immunoblot analyses were performed as previously described [19 (link)]. Briefly, N-ethylmaleimide (10 mM, Sigma) was added to the radioimmunoprecipitation assay (RIPA) buffer (Upstate Biotechnology). The lysates were incubated with the indicated antibodies and Protein G agarose at 4°C for 12 h, and the beads were washed three times with cold RIPA buffer. Immunoblot analysis was performed as previously described [19 (link)]. Primary antibodies were obtained from the following sources; anti-pan-Ras (Millipore); -K-Ras, -N-Ras, -H-Ras, -β-actin, -p-ERK, -p-AKT, -PKCδ, -PKCα,-PCNA, and -c-Myc (Santa Cruz Biotechnology); -HA, -PKCε, and -PKCζ (Cell Signaling Technology); -Flag (Sigma); and -ER-α36 (Cell Applications). Secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse (Cell Signaling Technology) and HRP-conjugated anti-rabbit (Bio-Rad).

Total protein was extracted from HL‐60 cells and mice tumours, and protein concentration was quantified by the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The protein samples were separated by 7.5%‐12% SDS‐PAGE gels (Applygen, Beijing, China) and transferred onto polyvinylidene fluoride membranes (120 minutes at 100 V) using standard procedures. The membranes were blocked in TBST (PBS containing 0.1% Tween) containing 5% non‐fat dry milk for 120 minutes at room temperature and then incubated with primary antibodies against CRT (1:8000; Abcam), C/EBPα (1:500; Abcam), PERK (1:1000; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), p‐PERK (1:1000; Santa Cruz Biotechnology Inc), AKT (1:8000; Abcam), p‐AKT (1:1000; Santa Cruz Biotechnology Inc), and GAPDH (1:1000; Santa Cruz Biotechnology Inc) overnight at 4°C. After washing with TBST, membranes were incubated with secondary antibody (1:3000; Anti‐rabbit IgG, HRP‐linked Antibody; Abcam) for 1 hour at room temperature. Membranes were then extensively washed and developed using an enhanced chemiluminescence detection system (CWC, Beijing, China).