This is a knockout-validated antibody summary, based on publications "FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c" [1], "FOXO1 couples metabolic activity and growth state in the vascular endothelium" [2], "Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy" [3], "A mutant allele encoding DNA binding-deficient FoxO1 differentially regulates hepatic glucose and lipid metabolism" [4], "Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin's glucose-lowering effects" [5], "Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple FoxO-deficient mice" [6], "The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help" (figure 1b, Western blot) [7] and "Genetic Ablation of FOXO in Mice to Investigate Its Physiological Role" (immunohistochemistry and Western blot, figure 2a and 3) [8]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Company: Cell Signaling Technology

Antibody: FoxO1

Catalog number: 2880

Summary: Rabbit monoclonal antibody raised against a GST-fusion peptide corresponding to the carboxy-terminal residues of human FoxO1. It detects endogenous levels of total FoxO1 protein and does not detect exogenously expressed family members FoxO3a or FoxO4. It reacts to human, mouse, rat, and monkey has been successfully used in western blot, immunoprecipitation, immunohistochemistry (paraffin), immunocytochemistry and flow cytometry.

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Figure 1. Western blot of Fox0 on total cell lysates from FoxO knock-out mice and littermates. HMGB1 is the loading control. From figure 1 [1].

WB, western blot

Liver extracts were prepared from FoxO knock-out mice and littermates and analyzed by Western blotting with the high-mobility group protein-1 (HMGB1) as loading control.

SuperSignal West Femto maximum sensitivity substrate (Pierce)

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Figure 2. Immunofluorescence staining for FOXO1 in a P5 mouse retina of 4-OHT-injected control and Foxo1 iEC-KO mice. From figure ED1 [2].

IHC, immunohistochemistry

P5 mouse retina of 4-OHT-injected control and Foxo1 iEC-KO mice. whole mouse eyes were fixed in 4% paraformaldehyde (PFA) on ice for 1h. Eyes were washed in PBS before the retinas were dissected and partially cut into four leaflets

blocking/permeabilization in 2% goat serum (Vector Laboratories), 1% BSA and 0.5% Triton X-100 (in PBS) for 1h at room temperature

incubated at 4°C overnight in incubation buffer containing 1% goat serum, 0.5% BSA and 0.25% Triton X-100 (in PBS) and 1:100 primary antibody

Alexa-Fluor 488-, Alexa-Fluor 555- or Alexa-Fluor 647-conjugated secondary antibodies (Invitrogen, 1:400) for 2h at room temperature

flat-mounted with Vectashield (Vector Laboratories) and examined by confocal laser microscopy (Leica TCS SP5 or SP8)

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Figure 3. From figure 1 in [3]. Western blot of skeletal muscle lysates from wild-type and muscle-specific triple FoxO1, 3, 4 knockout mice.

Catalog number is 2880 (Clone C29H4), from kit 9946.

Western blot

Skeletal muscle lysates from wild-type mice and mice with muscle-specific triple knockout of FoxO1, 3 and 4.

Samples contain small numbers of contaminating endothelial, fibroblasts, macrophages and blood cells which will not be deleted for FoxO1, 3, 4 and will likely give rise to a background signal.

Anti-FoxO1 diluted 1:1000.

Super Signal West Pico ECL reagent (Pierce).

Western blot | Immunohistochemistry

Islet extracts of 24-week-old db/db, WT (C57BL/6) and FoxO-KO. For IHC, 5-μm-thick paraffin sections were used.

FITC-, Cy3-, and Alexa-conjugated donkey secondary antibodies (Jackson Immunoresearch Laboratories, and Molecular Probes).

Please see Figure 1b,c in the article [6].

Western blot

Livers from control, FoxO1−/− and FoxO1−/dbd mice. Frozen livers were homogenized in ice-cold lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% glycerol, 2% NP-40, 1 mmol/L EDTA, 20 mmol/L NaF, 30 mmol/L Na4P2O7, 0.2% [w/v] SDS, and 0.5% [w/v] sodium deoxycholate) supplemented with protease/phosphatase inhibitors (Cell Signaling Technology).

Western blot of liver extracts from fasted control, FoxO1−/− and FoxO1−/dbd mice. Please see Figure 1e in the article [4].