Ecl plus chemiluminescence kit

Brain tissues were collected and stored at −80 °C after being perfused with cold PBS (0.1 M, pH 7.4) at 12 h, 1 day, 3 days, 5 days, 7 days for the time course study, and at 3 days for the mechanistic study after GMH induction, respectively. Western blot was performed as previously described [25 (link)]. After sample preparation, 50 μg protein per sample was loaded onto 10–12% SDS-PAGE gels, ran for 90 min at 100 V, and was transferred onto 0.2 mm or 0.45 mm nitrocellulose membranes at 100 V for 120 min (Bio-Rad) [24 (link)]. The membranes were blocked for 2 h in 5% non-fat milk in Tris-buffered saline with 0.1% tween20, followed by overnight incubation at 4 °C with the following primary antibodies: anti-human NT-4 (ThermoFisher, USA), anti-NT-4 (Santa Cruz, USA), anti-TrkB (Abcam, USA), anti-pTrkB (ThermoFisher, USA), anti-PI3K(Abcam, USA), anti-Akt (Abcam, USA), anti-pAkt (Abcam, USA), anti-FoxO1(CST, USA), and anti-IL6 (Santa Cruz, USA). The same membranes were probed with actin (Santa Cruz, USA) as internal loading controls. Appropriate secondary antibodies (Santa Cruz, USA) were incubated with membranes for 2 h at room temperature. Bands were visualized using ECL Plus Chemiluminescence kit (Amersham Biosciences, USA) and quantified through ImageJ 4.0 (Media Cybernetics). And we have the gel scans of all proteins in this study (Supplementary Fig. 3).

Infected cells were harvested in ice-cold PBS containing 1 mM Na₃VO₄ (Sigma-Aldrich). Western blotting was done as previously described [22] (link). Rabbit α-CagL antiserum was raised against the C-terminal peptide (C-RSLEQSKRQYLQER) of the protein and was prepared by Biogenes (Berlin, Germany). The α-HA-tag antibody (NEB Cell Signaling, Frankfurt/M., Germany) was also used to detect tagged CagL. The pan-α-phosphotyrosine antibody PY-99 (Santa Cruz) and α-CagA (Austral Biologicals, San Ramon, CA, USA) were used to investigate the phosphorylation of CagA [23] (link). The α-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz) served as loading control in each Western blot (data not shown). As secondary antibodies, horseradish peroxidase conjugated α-mouse, α-rabbit, or α-goat polyvalent sheep immunoglobulin were used and antibody detection was performed with the ECL Plus chemiluminescence kit (Amersham Pharmacia Biotech) [24] (link). Band intensities were quantitated with the Lumi-Imager F1 (Roche Diagnostics, Mannheim, Germany) [25] . The data are representative from three independent experiments.

A dose-dependent study on the caspase-8, -9 and -3/7 activation was carried out using Caspase-Glo^® 3/7, 8 and 9 kit (Promega, Madison, WI, USA). In brief, a total of 5 × 10³ A549 cells were seeded per well in a white 96-well microplate and incubated with different concentrations of AMEAE for 24 h. Then, caspase-Glo reagent (100 μl) was added to the cells for 30 min. The induced activation of caspases was measured using a Tecan Infinite^®200 Pro (Tecan, Männedorf, Switzerland) microplate reader.
To determine the protein expression of cleaved caspase-3 and -9, western blot analysis was carried out as previously described in detail [19 (link)]. In brief, A549 cells treated with vehicle DMSO or AMEAE at different concentrations were washed with PBS and lysed in ice-cold Radio Immuno Precipitation Assay (RIPA) buffer. Cell extracts (80 μg protein) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to nitrocellulose membrane, probed with anti-β-actin, anti-cleaved caspase-9 and anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated secondary antibodies were used followed by the detection of protein expression using the ECL plus chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA).

MRA and skeletal muscles from both ischemic and non-ischemic legs of each rat were collected separately, frozen and then homogenized. Proteins were separated by SDS-PAGE. In the ischemic and non-ischemic muscle, eNOS, phospho-eNOS, caveolin-1, Akt, Phospho-Akt (P-Akt), PI3K, hypoxia-inducible factor 1 alpha (HIF-1α) and VEGF were then visualized with ECL-Plus Chemiluminescence kit (Amersham, United Kindom).
Antibodies directed against eNOS (1:500; BD 610297), anti-P-eNOSser1177 (1:500; BD 612392) and caveolin-1 (1:1000, BD 610407) were purchased from BD Transduction Laboratories (United States); Akt (1:1000, CellSignaling #9272), and P-Akt-ser473 (1:1000, CellSignaling #9271) antibodies were from Cell signaling (United States); and PI3K (p85α 1:500; sc-1637) and VEGF (1:500; sc-7269) antibodies from Santa Cruz (United States).