Immunohistochemistry was performed using polyclonal antibody for SphK1 and COX-2. Tissue samples were recovered, fixed in 10% buffered formalin, and embedded in paraffin. Section (4 µm) were dewaxed and hydrated through graded ethanol, cooked in 25 mM citrate buffer, pH 6.0, in a pressure cooker for 10 min, transferred into boiling deionized water, and let to cool for 20 min. Tissue sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity. The slides were incubated with anti-SphK1 (Abcam) and anti-COX-2 (Cell Signaling) antibodies at a working dilution of 1:200 overnight at 4 °C [18 (link)] and a secondary polymer-based detection system (EnVision+ System Labelled Polymer-horseradish peroxidase anti-rabbit; Dako). The chromogen was 3,3′diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA, USA) and sections were counterstained with haematoxylin. The specificity of technique was evaluated by negative controls (omitting the incubation with the primary antibody and incubating it with nonimmune sera). Areas of staining were analyzed by WinRoof version 6.3 (Mitani, Tokio, Japan) software with ten non-overlapping randomly chosen histological fields. Results were expressed as the percentage of stained cells in each field.
Anti sphk1
Manufactured by Abcam
Sourced in China, United States
Anti-SphK1 is an antibody that binds to and detects Sphingosine kinase 1 (SphK1), an enzyme involved in the production of the lipid signaling molecule sphingosine-1-phosphate. This antibody can be used for research purposes to study the expression, localization, and regulation of SphK1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti sphk2, by Abcam (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Anti cox 2, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti cyclophilin, by Abcam (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti sgpl1, by Santa Cruz Biotechnology (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Anti lyve 1, by Abcam (1 mentions)
Anti ck8, by Abcam (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Tcs sp2 aobs confocal laser scanning microscope, by Leica (1 mentions)
Bx41 light microscope, by Olympus (1 mentions)
Anti cd31, by BD (1 mentions)
Saponin from quillaja bark, by Merck Group (1 mentions)
7 protocols using anti sphk1
1
Immunohistochemical Analysis of SphK1 and COX-2
2
Protein Expression Analysis via Western Blot
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For SGPL1, SPHK1, SPHK2, twenty-five micrograms of total protein lysate were immunoblotted with the following antibodies: anti-SGPL1 (1:1000) (Santa Cruz, Cat. N. sc-67368), anti-SPHK1 (1:1000) (Abcam, Cat. N. ab71700; lot numbers: GR17790-4 and GR17790-24) and anti-SPHK2 (1:1000) (Abcam, Cat. N. ab37977; lot numbers: GR31063-12 and GR31063-39). For phospho-AKT, AKT, phospho-ERK and ERK, total lysate was immunoblotted with the following antibodies: anti-phospho-AKT (1:1000) (Cell Signaling, Cat. N. 9271), anti-AKT (1:1000) (Cell Signaling Cat. N. 2920), anti-phospho-ERK (1:1000) (Cell Signaling Cat. N. 9101), anti-ERK (1:1000) (Cell Signaling Cat. N. 9102), and anti-ERK (1:1000) (Cell Signaling Cat. N. 4696). For protein normalization, anti-Actin (1:5000) (Sigma Aldrich, Cat. N. A5441) or anti-Cyclophilin (1:3000) (Abcam, Cat. N. ab16045) was used. Protein bands were detected by ECL Prime (GE Healthcare) and quantitated with Quantity One Software (Bio-Rad Laboratories).
3
Immunohistochemical Analysis of Tumor Markers
4
Immunostaining of mESCs for SPHK1, SPHK2, and NANOG
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Immunostaining was performed by fixing mESCs in Millicell glass EZ slide (Millipore) with 4% paraformaldehyde (Sigma) at room temperature (RT) for 20 minutes. Cells were blocked for an hour by incubating in phosphate‐buffered saline (PBS) supplemented with 10% FBS, 0.1% IgG‐free bovine serum albumin (BSA, Jackson ImmunoResearch, West Grove, Pennsylvania) and 0.1% saponin from quillaja bark (Sigma) at RT. Cells were incubated overnight at 4°C with anti‐SPHK1 (Abcam, Cambridge, UK, 1:50) or anti‐SPHK2 antibody (Abcam, 1:50) in blocking buffer. Fluorescence‐conjugated secondary antibody was used for visualization. Nuclei were labeled with 4′,6‐diamidino‐2‐phenylindole (Molecular Probes, Invitrogen, Carlsbad, California). Images were collected on a Zeiss (Oberkochen, Germany) LSM 800 confocal microscope with ZEN software. To measure NANOG, the same protocol was used except that mESCs were fixed in a tissue culture dish, stained with anti‐NANOG antibody (Abcam, 1:100) and visualized on a Zeiss epifluorescence microscope with the AxioVision software.
5
Immunoblotting Analysis of Sphingosine Kinases
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Cells were lysed (20 min at 4°C) in Tris-buffered saline (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM PMSF, and protease inhibitors. After centrifugation, supernatants were assayed for proteins [26 (link)], and equal amount of proteins (15–30 μg) were submitted to SDS-PAGE on 10% polyacrylamide gel. After transfer onto nitrocellulose membranes, samples were incubated (overnight, 4°C) with the following antibodies: anti-SphK1 and anti-SphK2 (Abcam, Cambridge, UK), anti-phospho-Akt, anti-caspase-3 antibody (Cell Signaling Technology, Inc., Danvers, MA), and then with an anti-HRP-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-β-actin (Sigma Aldrich, St. Louis, MO) and anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used as loading control. Immunoreactive signals were visualized by Supersignal West Femto (Thermo Scientific (Rockford, IL), and exposure to Kodak Biomax film (Rochester, NY). Immunoreactive band density was determined with a densitometer (Bio-Rad Laboratories, Hercules, CA; Quantity One software).
6
HMGB1 Acetylation Regulation in LPS-Induced Inflammation
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LPS (Escherichia coli 0111: B4), SKI-5C, K6PC-5, and glycyrrhizin were purchased from Sigma-Aldrich (Shanghai, China). Gadolinium chloride (GdCl3) was obtained from Absin (Shanghai, China). Collagenase IV, protein interaction pull-down kit, anti-CD68, anti-phospho-HDAC4, anti-calcium/calmodulin protein kinase II-δ (CaMKII-δ), and anti-phospho-CaMKII-δ were obtained from Thermo Scientific (Shanghai, China). Anti-SphK1 and Anti-HMGB1 (ChIP Grade) were obtained from Abcam (Shanghai, China). Anti-HMGB1, anti-GAPDH, anti-Lamin B1, anti-histone H3, anti-E1A-associated protein p300 (p300), anti-CREB-binding protein (CBP), anti-p300/CBP-associated factor (PCAF), anti-histone deacetylase (HDAC) 1, anti-HDAC4, and anti-acetylated-lysine were purchased from Cell Signaling Technology (Shanghai, China). Anti-HMGB1 (Acetyl-Lys12) was obtained from Aviva Systems Biology (San Diego, CA, USA). Anti-HA and anti-His were purchased from Zoonbio Technology (Nanjing, China). The manufacturers of all the antibodies used in this study are listed in Supplementary Table 1 .
7
Protein Extraction and Immunoblotting for SPHK2 and STAT Signaling
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Total proteins were extracted using ice-cold lysis buffer (1% NP-40, 1mM DTT, phosphatase inhibitor and protease inhibitor cocktail in PBS) followed by immunoblotting [57 (link),58 (link)]. Primary antibodies against SPHK2, pSTAT1 (Y701), STAT1, IRF9, pSTAT2 (Y690), and STAT2 were purchased from Cell Signalling Technologies, USA. Anti-phosphorylated SPHK1 (S225) was obtained from ThermoFisher Scientific, Waltham, Massachusetts, USA; anti-SPHK1 from Abcam, USA; and anti-GAPDH from Santa Cruz Biotechnology, Santa Cruz, CA, USA. All images were captured using the ChemiDocTM XRS+ molecular imager (Bio-Rad Laboratories, Hercules, CA, USA).
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