Hrp conjugated second antibody

RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) were used to extract the proteins from cells. Protease (Thermo Fisher Scientific) and phosphatase inhibitors (Roche, Basel, Switzerland) were added in the buffer to inhibit the activations of Protease and phosphatases. Then the proteins were heated with 4× loading buffer at 95 °C, 10 min. After detecting the protein concentrations by the Micro BCA Protein Assay Kit (Thermo Fisher Scientific), we separated twenty micrograms of total protein lysate by SDS-PAGE and electro-transferred onto a polyvinyl difluoride membrane. The blots were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% milk or 5% BSA for 1 h at room temperature, then incubated with primary antibodies against pAMPK, p-AKT, total AKT, pSMAD3, SMAD3, SMAD4, aH3, H3 (Abcam) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Subsequently, the membranes were washed with TBST for three times and incubated with HRP-conjugated second antibodies (Santa Cruz) for 1 h at room temperature. The immune-reactive bands were detected by an Immobilon Western Chemiluminescent HRP Substrate kit (Merck Millipore). Pictures of the bands were recorded by Amersham Imager 600 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). All the western blot experiments were repeated at least three times.

The hippocampus was homogenized in ice-cold lysis buffer containing protease inhibitor cocktails. Equal protein amounts were electrophoresed on a 10% SDS-PAGE gel and subsequently transferred to PVDF membranes. The membrane was blocked with 5% nonfat milk for 1 h at 24°C, followed by incubation overnight at 4°C with primary antibodies to RvD 1 receptor FPR2 (Thermo Fisher Scientific, Rockford, IL, United States), proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 (Abcam, Cambridge, MA, United States), and β-actin (Santa Cruz Biotechnology, CA, United States). Blots were then incubated with HRP-conjugated second antibodies (Santa Cruz Biotechnology, CA, United States) for 1 h. The immunoreactive bands were visualized with ECL Plus reagents (Amersham Biosciences Inc. Piscataway, NJ) and developed on a film. Band density measurements were made using ImageJ software e (NIH, Bethesda, MD, United States).

Protein levels of pro-inflammatory cytokines in the hippocampus were analyzed by western blot as previously described (Zhang et al., 2016 (link)). Briefly, the hippocampus was quickly dissociated from brain and homogenized in an ice-cold lysis buffer with protease inhibitor. The protein concentration was determined by a Bradford assay using BSA as the standard. Samples were electrophoresed on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the gels were transferred to polyvinylidene difluoride (PVDF) membranes. After blocking for 1 h in 5% non-fat dry milk, the membranes were incubated with primary antibodies to tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States) at 4°C overnight. Membranes were then washed and incubated at room temperature with HRP-conjugated second antibody (Santa Cruz Biotechnology, Inc.) for 1 h. The enhanced chemiluminescence detection system (Amersham) was applied to visualize the immunoreactive bands, and the band densities were analyzed with ImageJ software (National Institutes of Health). All data were normalized by β-actin.

The total, cytoplasmic and nuclear proteins were isolated using protein extraction kit (Beyotime) according to the manufacturer’s instructions. After quantitation (BCA kit, Beyotime), protein was separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred onto PVDF membrane. Antibodies against β-actin, β-catenin, smooth muscle myosin heavy chain protein (SM-MHC), Notch1 (Santa Cruz, CA, USA), GAPDH, histone H3, phospho-p38, phospho-ERK1/2 (Cell Signaling Technology, MA, USA) and smooth muscle α-actin (SM α-actin) (Sigma) were used to probe with the interest blots. Finally, protein expression was detected with HRP-conjugated second antibody (Santa Cruz) and ECL (Pierce, IL, USA) luminescence method.

After brain tissues were homogenized with 1 mM EDTA and 2.5 mL cell lysate, 70 μg of proteins was separated on 12% SDA-PAGE and electrophoretically transferred to PVDF membranes (Pall corporation, NY, USA). The blots were incubated with specific primary antibody against caspase-3 and p53 (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. HRP-conjugated second antibody (Santa Cruz Biotechnology, Inc., Dallas, USA) was used for further incubation for 1 h. The blots were washed with TBST. Signals were detected by an enhanced chemiluminescence detection system (Millipore, St. Louis, MO, USA) and analyzed by Image-Pro Plus 7.0 software. Targeted bands were normalized to β-actin (KeyGEN Biotech, Nanjing, China) to ensure equal protein loading.