Treg cells were isolated from WT and SGK1-deficient were stimulated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) on chamber slides at 37° C for 20 min. After fixation with 4% paraformaldehyde, cells were permeabilized with Foxp3 Fixation/Permeabilization buffer (eBioscience) according to the manufacturer’s instructions. After blocking with Permeabilization buffer and 3% BSA, cells were incubated with 1:250 diluted mouse anti-Foxo1 (ab1102, Abcam), rat anti-Foxp3 (FJK-16 s, eBioscience), and rabbit anti-SGK1 (ab43606, Abcam), followed by Alexa555-anti-mouse, allophyco-cyanin (APC)-anti-rabbit and fluorescein isothiocyanate (FITC)-anti-rat secondary antibodies in Permeabilization buffer and 1% BSA. Slides were mounted with gold anti-fading mounting buffer (Invitrogen). Images were acquired with a Leica TCS SP5-II confocal microscope. For quantitative analysis, five fields were selected randomly and total cells in the field were manually counted and grouped with Volocity software (PerkinElmer), on the basis of their Foxo1 nuclear or cytosolic localization.
Ab43606
Manufactured by Abcam
Ab43606 is a primary antibody that binds to a specific target. It can be used for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 2 confocal microscope, by Leica (2 mentions)
Fjk 16, by Thermo Fisher Scientific (2 mentions)
Foxp3 fixation permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Volocity software, by PerkinElmer (2 mentions)
Ab1102, by Abcam (2 mentions)
Gold anti fading mounting buffer, by Thermo Fisher Scientific (2 mentions)
Ab22897, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Alexa 488 a11008, by Thermo Fisher Scientific (1 mentions)
Alexa 594 a11042, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Ab49822, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab32117, by Abcam (1 mentions)
Keratinase, by Merck Group (1 mentions)
Keratinase 2, by Merck Group (1 mentions)
Ab32370, by Abcam (1 mentions)
Bb 3501, by Cytiva (1 mentions)
Ab8245, by Abcam (1 mentions)
4 protocols using ab43606
1
Foxo1, Foxp3, and SGK1 Localization in Treg Cells
2
Foxo1, Foxp3, and SGK1 Localization in Treg Cells
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Treg cells were isolated from WT and SGK1-deficient were stimulated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) on chamber slides at 37° C for 20 min. After fixation with 4% paraformaldehyde, cells were permeabilized with Foxp3 Fixation/Permeabilization buffer (eBioscience) according to the manufacturer’s instructions. After blocking with Permeabilization buffer and 3% BSA, cells were incubated with 1:250 diluted mouse anti-Foxo1 (ab1102, Abcam), rat anti-Foxp3 (FJK-16 s, eBioscience), and rabbit anti-SGK1 (ab43606, Abcam), followed by Alexa555-anti-mouse, allophyco-cyanin (APC)-anti-rabbit and fluorescein isothiocyanate (FITC)-anti-rat secondary antibodies in Permeabilization buffer and 1% BSA. Slides were mounted with gold anti-fading mounting buffer (Invitrogen). Images were acquired with a Leica TCS SP5-II confocal microscope. For quantitative analysis, five fields were selected randomly and total cells in the field were manually counted and grouped with Volocity software (PerkinElmer), on the basis of their Foxo1 nuclear or cytosolic localization.
3
Immunodetection of SGK1.1, Kv7.2, and Nedd4-2
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For SGK1.1 detection we used a rabbit polyclonal antibody produced in our laboratory (Martin-Batista et al., 2021 (link)). SGK1 was detected using rabbit anti-SGK1 polyclonal antibody (Abcam, ab43606). Kv7.2 was detected with rabbit anti-Kv7.2 polyclonal antibody (Abcam, ab22897). YFP-tagged SGK1.1 and GFP-tagged Kv7 subunits were detected using mouse anti-GFP monoclonal antibody (Abcam, ab290). Nedd4-2 was detected with a rabbit polyclonal antibody from Cell Signaling (4013S). GAPDH was detected with a mouse monoclonal antibody (Abcam, 9484). Secondary antibodies conjugated to fluorophores were obtained from Thermo Fisher Scientific (Alexa 594, A-11042; Alexa-488, A-11008).
4
Western Blot Analysis of Chondrocyte Proteins
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Cartilage tissue was ground and cell samples were washed with precooled PBS and were lysed in a cold radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Beijing, China). Protein concentration was detected using bicinchoninic acid (BCA) protein assay kit (Applygen, Beijing, China). All samples were treated with chondroitin enzymes ABC, keratinase, and keratinase II (Sigma-Aldrich) without the protease. Next, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a membrane of polyvinylidene fluoride (Thermo Fisher Scientific), and then blocked at room temperature. The membrane was probed with diluted primary antibodies to GAPDH (1:1000, ab8245, Abcam), SGK1 (ab43606), HDAC2 (Y461, ab32117), Bcl-2 associated X protein (Bax; E63, ab32503), B cell lymphoma-2 (Bcl-2; E18, ab32370), and Cleaved Caspase-3 (ab49822) overnight at 4 °C. The 1-h culture of cells was conducted after supplementing with secondary antibody labeled by horseradish peroxidase (HRP). Bands of protein were visualized using the enhanced chemiluminescence (BB-3501, Amersham-Pharmacia Biotech, Freiburg, Germany). Optical density of image was determined using ImageJ Software, with GAPDH as the internal reference.
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