Gure 9 (a) Representative photos of AOPPs immunochemistry in paraffin sections of resected intestinal specimens

Gure 9 (a) Representative photos of AOPPs immunochemistry in paraffin sections of resected intestinal specimens from individuals with CD (n 23). Standard tissue adjacent for the diseased intestine was used as a normal control. (b) Immunofluorescence TUNEL labeling in small intestinal epithelium sampled from individuals with CD. (c) The higher AOPPs immunoreactivity score revealed an improved CETP Inhibitor Gene ID quantity of apoptotic cells. HPF: high-power fields. Po0.05 versus controlApoptosis assays in IEC-6 cultures. Assessment of FITC annexin V-labeled apoptotic cells was performed according to the protocol provided by the manufacturer (Becton Dickinson, Franklin Lakes, NJ, USA). Cells had been seeded on six-well plates and treated with or without AOPP-RSA for the indicated time; cells (1 106) were suspended in buffer containing FITC annexin V and PI. The samples had been analyzed having a FACS Calibur flow cytometer (Becton Dickinson). A total of 10 000 cells were analyzed per determination. Cells were viewed as apoptotic if they were undergoing either early (Annexin-V-positive, PI-negative) or late apoptosis (Annexin-V-positive, PI-positive). Determination of ROS generation. Intracellular ROS generation was measured using a flow cytometer (Becton Dickinson) with all the probe DCFH-DA (20 ,70 -DCF-diacetate), which is a cell-permeable, non-fluorescent dye that may be oxidized to the fluorescent 20 ,70 -DCF by ROS inside cells. Briefly, IEC-6 cultures have been incubated with 10 mM DCFH-DA for 30 min at 37 1C followed by AOPPs therapy as described above. Western blotting. Cultured cells or frozen rat intestinal tissue samples were lysed in radio-immunoprecipitation assay buffer, and protein was collected following centrifugation and mixed with 5 sodium dodecyl sulfate (SDS) sample buffer. The samples had been separated by SDS-polyacrylamide gel electrophoresis (Page) applying 82 acrylamide gels and after that FP Compound transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Immediately after incubation with major and secondary antibodies, the protein bands had been detected with chemiluminescence detection reagents (Millipore). The following antibodies (Abs) have been used: goat anti-p22phox, goat anti-gp91phox pAb, and goat anti-p47phox pAbs had been all from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-PARP-1 pAb, antiBcl-2 pAb, anti-Bax pAb, anti-caspase-3 pAb, anti-JNK Ab, and anti-pJNK Ab have been Cell Death and Illness from Cell Signaling Technologies (Beverly, MA, USA); anti-PAR mAb was from Millipore; rabbit anti-P47phox pAb was from Sigma; and anti-AIF Ab was from Abcam (Cambridge, UK). Mouse anti-AOPP Ab was a gift from professor Fu Ning (Southern Medical University, Guangzhou, China). Mouse anti-b-actin Ab and goat anti-mouse, rabbit anti-goat, and goat anti-rabbit IgG-horseradish peroxidase (HRP) had been bought from Boster (Wuhan, China). p47phox phosphorylation. p47phox phosphorylation in IEC-6 cultures was measured by immunoprecipitation as described previously.18 Briefly, cell lysates were incubated with protein A/G agarose beads (Santa Cruz Biotechnology), and also a polyclonal rabbit anti-phosphoserine Ab (Abcam). The precipitated immunocomplexes have been resolved by SDS-PAGE, transferred onto PVDF membranes (Millipore), incubated with an HRP-conjugated rabbit anti-p47phox antibody (Sigma), and subjected to chemiluminescence detection as described above. Immunofluorescence staining. p47phox translocation in the cytoplasm for the membrane and AIF migration had been detected employing immunofluor.