Anti-SGK1 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SGK1 antibody (AB43606)

ICC/IF image of ab43606 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab43606 at 5μg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• WB

Ap19106****

Western blot - Anti-SGK1 antibody (AB43606)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab43606 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-SGK1 antibody (ab43606) at 1 µg/mL

Lane 1:

Human BT549 (Breast carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Western blot - T-47D whole cell lysate (<a href='/en-us/products/cell-lysates/t-47d-whole-cell-lysate-ab14899'>ab14899</a>) at 10 µg

Lane 3:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Predicted band size: 48 kDa

Observed band size: 49 kDa

true

Exposure time: 1min

• WB

Ap19106****

Western blot - Anti-SGK1 antibody (AB43606)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab43606 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-SGK1 antibody (ab43606) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 4:

Human pancreas tissue lysate - total protein (<a href='/en-us/products/unavailable/human-pancreas-tissue-lysate-total-protein-ab29816'>ab29816</a>) at 10 µg

Lane 5:

Lung (Human) Tissue Lysate at 10 µg

Lane 6:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Predicted band size: 48 kDa

Observed band size: 38 kDa,49 kDa,51 kDa,60 kDa,95 kDa

true

Exposure time: 5s

• WB

CiteAb

Western blot - Anti-SGK1 antibody (AB43606)

SGK1 western blot using anti-SGK1 antibody ab43606. Publication image and figure legend from Evans, R. D. R., Antonelou, M., et al., 2020, Nat Commun, PubMed 32868758.

ab43606 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab43606 please see the product overview.

Expression of the components of the intracellular pathway that mediate salt-driven IL-17 inflammation and salt rescue of in vitro IL-17 responses in salt-losing tubulopathy patients.a IL-23 receptor expression on unstimulated CD4+ cells in healthy controls (n = 8) and SLT patients (n = 7) : HC 0.42% (0.34–1.01), SLT 0.79% (0.73–2.60), p = 0.07. Groups are compared with a two-sided Mann–Whitney test. Error bars represent interquartile range around the median. b IL-23 receptor expression on unstimulated CD4− cells in healthy controls (n = 8) and SLT patients (n = 7) : HC 3.34% (2.26–6.56), SLT 9.67% (5.64–10.65), p = 0.02. Groups are compared with a two-sided Mann–Whitney test. Error bars represent interquartile range around the median. c Relative lymphocyte NFAT5 expression in healthy controls (n = 13) and SLT patients (n = 11) : HC 1.01 AU (0.70–1.95), SLT 1.10 AU (0.76–1.62), p = 0.85. Groups are compared with a two-sided Mann–Whitney test. Error bars represent interquartile range around the median. d Relative lymphocyte SGK1 expression in healthy controls (n = 7) and SLT patients (n = 13) : relative intensity HC = 1.07 AU (0.73–1.2), SLT = 1.09 AU (0.80–1.31), p = 0.70. Groups are compared with a two-sided Mann–Whitney test. Error bars represent interquartile range around the median. e Representative western blot of SGK1 expression in HCs and SLT patients. SGK1 expression was determined in SLT patients (n = 13) and healthy controls (n = 7) across three experiments. f Th17 polarisation in HC (n = 27) and SLT patients (n = 25) in standard media, and in SLT patients in media supplemented with 40 mM NaCl : HC + 0 mM NaCl 3.2% (2.5–6.3); SLT + 0 mM NaCl 1.6% (0.8–2.0); SLT + 40 mM NaCl 3.1% (2.4–5.3); p = <0.0001. Groups are compared using a Kruskal–Wallis test with Dunn’s multiple comparison testing (shown with significance bars). Error bars represent interquartile range around the median. g Tc17 polarisation in HC (n = 27) and SLT patients (n = 25) in standard media, and in SLT patients in media supplemented with 40 mM NaCl : HC + 0 mM NaCl 1.5% (0.6–3.0); SLT + 0 mM NaCl 0.6% (0.3–1.1); SLT + 40 mM NaCl 1.6% (0.9–3.1); p = 0.0007; groups are compared using a Kruskal–Wallis test with Dunn’s multiple comparison testing (shown with significance bars). Error bars represent interquartile range around the median. ns not significant (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. HC healthy control, SLT salt-losing tubulopathy, AU arbitrary units. Source data are provided as a Source data file.

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