Approval from the Roswell Park Cancer Institution Animal Care and Use Committee was obtained for all experiments based on our extensive experience in animal models (32 (link)–36 ). Female C57BL/6 mice and B6.cg-Lepob (OB/OB) mice were obtained from Jackson Laboratory. OB/OB mice were fed a high fat diet (Envigo, TD.88137) from 10 days prior to implantation of cancer cells in the obesity model. 1×106 of E0771 breast cancer cells were inoculated into the #2 mammary fat pad under direct vision as described (13 (link), 37 (link)–41 ). Mice were treated with doxorubicin and/or FTY720. Doxorubicin was administrated by i.p. at a dose of 5 mg/kg on Day 0 and 3. FTY720 was administered everyday by gavage at a dose of 1 mg/kg. Anti-SphK1 antibody (Abcam) was used at 1:100 dilutions for immunohistochemistry.
Anti sphk1 antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-SphK1 antibody is a laboratory reagent used for the detection and analysis of Sphingosine kinase 1 (SphK1) protein in various biological samples. It is a specific antibody that binds to SphK1, allowing for its identification and quantification through techniques like Western blotting, immunohistochemistry, and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Td 88137, by Inotiv (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Actin c 2, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
3 protocols using anti sphk1 antibody
1
Modulating Obesity and Breast Cancer
2
Immunohistochemical Analysis of SphK1 in Colon Cancer
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Colon tumors used were obtained with patient consent and approved by the institutional review board at the National University Health System in Singapore. Immunohistochemical staining of SphK1 expression was performed on tissue microarrays containing 303 colon adenocarcinomas. Tissue microarrays were constructed according to previously published methods.18 (link) Tissue microarray slides were deparaffinized with xylene, and rehydrated in a graded alcohol series. Slides were then heated with the antigen unmasking solution (Vector Laboratories, Peterborough, UK) for 10 min, and stained with the DakoCytomation EnVision+ System–HRP (AEC) kit (Dakocytomation, Singapore, Singapore). After blocking endogenous peroxidase with hydrogen peroxide, slides were incubated with the primary rabbit anti-SPHK1 antibody (Abcam, Cambridge, UK) and secondary anti-rabbit antibody. Tissue sections were counterstained with hematoxylin. A four-tier scoring system was used, whereby the intensity of SphK1 expression was classified on an increasing scale of 0–3, with “0” being undetectable and “3” being the most intensely stained. Figure 1a shows representative images of each staining intensity, and Table 1 shows the distribution of SphK1 staining expression in the patient cohort.
3
Quantification of Phosphorylated Sphingosine Kinase 1
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Cell pellets were lysed with ice-cold RIPA lysis buffer supplemented with protease inhibitor (Thermo scientific #A32965) and Phosphatase inhibitor (Thermo scientific #A32957). Protein concentrations of the clarified lysates were determined with the DC Protein Assay (BioRad). 50ug of total protein from each sample were resolved on 10% SDS-PAGE. Gels were then transferred to nitrocellulose membrane and were immunoblotted for proteins of interest (SK1 and p-SK1). Actin was used for loading controls. The following antibodies were used: Actin (C-2) (Santa Cruz Biotechnology sc-8432), Anti-SPHK1 antibody (Abcam ab71700), and SPHK1-Phospho-Ser225 Antibody (Proteintech 19561–1-AP). Proteins of interest were visualized and quantified by the Li-Cor Odyssey Classic or CLx imaging system and the Image Studio software package.
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