Cells were fixed with cold 4% paraformaldehyde for 15 min and washed thrice in phosphate-buffered saline. Thereafter, they were blocked in 10% normal donkey serum for 1 h at RT, and incubated with anti-t- FoxO3a (Cell Signaling Technology) at 4 °C overnight. Subsequently, the cells were incubated with Cyanine 3 (Cy3)-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at RT. Nuclei were viewed with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) after 5-min incubation at RT. Cells were imaged using a Zeiss LSM700 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). The positive nuclear FoxO3a counting in each group was calculated based on 100% control
Anti t foxo3a
Manufactured by Cell Signaling Technology
Sourced in United States, Canada
Anti-t-FoxO3a is a laboratory reagent produced by Cell Signaling Technology. It is an antibody that detects the phosphorylated form of the transcription factor FoxO3a.
Automatically generated - may contain errors
Lab products found in correlation
Anti p foxo3a, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Cyanine 3 cy3 conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Ab3623, by Merck Group (1 mentions)
Anti bcl 2 sc 492, by Santa Cruz Biotechnology (1 mentions)
Anti active caspase 3, by Merck Group (1 mentions)
Anti t akt, by Cell Signaling Technology (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti tubulin, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti sod2, by Santa Cruz Biotechnology (1 mentions)
Anti rip3, by Santa Cruz Biotechnology (1 mentions)
Anti gpx4, by Abcam (1 mentions)
Anti il1 beta, by Abcam (1 mentions)
Anti p foxo1, by Cell Signaling Technology (1 mentions)
3 protocols using anti t foxo3a
1
Immunofluorescent Quantification of Nuclear FoxO3a
2
Molecular Mechanisms of Kidney Injury
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The following primary antibodies were used for immunoblot analysis or immunohistochemical staining: anti-KIM-1 (ab56015; Abcam, Cambridge, UK), anti-8-OHdG (MOG-100 P; JaICA, Shizuoka, Japan), anti-4-HHE (MHH-030n; JaICA), anti-MnSOD (ab16953; Abcam), anti-PI3K (610045; BD Transduction Laboratories, San Jose, CA, USA), anti-p-AKT (Ser473) (9271 S; Cell Signaling Technology, Danvers, MA, USA), anti-t-AKT (9272 S; Cell Signaling Technology), anti-p-FoxO3a (9466 S; Cell Signaling Technology), anti-t-FoxO3a (2497 S; Cell Signaling Technology), anti-β-actin (A5441; Sigma-Aldrich), anti-COX-IV (A301-899 A; Bethyl Laboratories, Montgomery, TX, USA), anti-Bcl-2 (sc-492; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax (DB005; Delta Biolabs, Gilroy, CA, USA), anti-active caspase-9 (9505 S; Cell Signaling Technology), and anti-active caspase-3 (AB3623; Millipore Corporation, St. Charles, MO, USA).
3
Quantification of Oxidative Stress Markers
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Whole cell or tissue samples were homogenized in RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX) with protease inhibitor cocktail (Thermo Scientific) and 40 μg total protein was used for immunoblot analysis according to our previous protocols [27] . Blots were probed with anti-Klotho (1:200; Thermo Scientific), anti-3-nitrotyrosine (1:500; Abcam, Cambridge), anti-SOD2 (1:500; Santa Cruz Biotechnology), anti-RIP1 (1:100; Santa Cruz Biotechnology), anti-RIP3 (1:500; Santa Cruz Biotechnology), anti-IL-1 beta (1:1000; Abcam), anti-tubulin (1:1000; Abcam), anti-beta-actin (1:2000; Abcam), anti-catalase (1:1000; Abcam), anti-GPX4 (1:1000; Abcam), anti-t-FoxO1 (1:1000; Cell Signaling Technology, Beverly, CA), antip-FoxO1 (1:1000; Cell Signaling Technology), anti-t-FoxO3a (1:1000; Cell Signaling Technology), anti-p-FoxO3a (1:1000; Cell Signaling Technology), or anti-GAPDH (1:1000; Cell Signaling Technology) antibodies overnight at 4 °C. After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; Cell Signaling Technology). After washing, specific signals were determined using an ECL kit (Thermo Scientific) on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai). Gray values were analyzed with ImageJ software.
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