Anti wnk4

Manufactured by Merck Group

Anti-WNK4 is a laboratory research tool used in the study of the WNK4 protein. WNK4 is a serine/threonine-protein kinase that plays a role in the regulation of ion homeostasis and blood pressure. Anti-WNK4 can be used to detect and quantify WNK4 expression in biological samples.

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Lab products found in correlation

Anti tubulin, by Merck Group (2 mentions) Anti flag, by Merck Group (2 mentions) Plasmamembrane isolation kit, by Invent Biotechnologies (2 mentions) Anti phospho spak osr1, by Merck Group (2 mentions) Anti spak, by Cell Signaling Technology (1 mentions) Ecl reagent, by PerkinElmer (1 mentions) Anti wnk1, by Cell Signaling Technology (1 mentions) Anti ubiquitin, by Cell Signaling Technology (1 mentions) Anti ncc, by Merck Group (1 mentions) Anti calcineurin a, by Cell Signaling Technology (1 mentions)

2 protocols using anti wnk4

Western Blot Analysis of Membrane Proteins

Cited 1 time

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Western blotting was performed as described previously [12 (link),18 (link)]. Plasma membrane fraction was purified using plasma
membrane isolation kit (Invent). Primary antibodies included anti-KLHL3 (Sigma)
[12 (link)], anti-Flag
(Sigma), anti-tubulin (Sigma), anti-WNK4 (produced in the lab; Supplemental Figure 2),
anti-phospho SPAK/OSR1 (Millipore) [19 (link)], anti-SPAK (Cell Signaling Technology; CST)
[19 (link)], anti-NCC
[12 (link)], anti-NCC
phosphorylated at Thr53 and Thr71 [20 (link)], anti-phospho PKC (bearing phosphorylation at Ser660 in
PKCβII; the antibody also detects phosphorylated PKCα,
βI, δ, ε, η, and θ; CST), anti-phospho
PKCα/β (bearing phosphorylation at Thr638/641; Abcam),
anti-total PKC, anti-phospho protein kinase A (PKA; bearing phosphorylation at
Thr197 in the catalytic domain), and anti-PKA catalytic domain (CST).

Ishizawa K., Xu N., Loffing J., Lifton R.P., Fujita T., Uchida S, & Shibata S. (2016). Potassium depletion stimulates Na-Cl cotransporter via phosphorylation and inactivation of the ubiquitin ligase Kelch-like 3. Biochemical and biophysical research communications, 480(4), 745-751.

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Quantification of Ubiquitylated WNK4 Protein

Cited 2 times

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Western blotting was performed as described (47 (link)). Plasma membrane fraction was purified using plasma membrane isolation kit (Invent biotechnologies). Enrichment in plasma proteins was confirmed in the laboratory (54 (link)). Equal amounts of protein were mixed with Laemmli sample buffer, separated on polyacrylamide gel, and transferred to nitrocellulose membrane. The membrane was incubated with primary and peroxidase-conjugated secondary antibodies, followed by imaging using ECL reagents (Perkin-Elmer). Tubulin and Ponceau S staining were used to ensure equal loading and transfer of samples. For the detection of ubiquitylated WNK4, WNK4-HA was purified by HA- immunoprecipitation (IP) from the cell lysates, and the ubiquitylated WNK4 was detected by Western blotting using anti-ubiquitin antibody (47 (link)). Monoclonal mouse antibody against KLHL3^S433-P was created in the laboratory as described (35 (link)) and was further characterized in this study. Rabbit polyclonal antibodies against NCC phosphorylated at T53 is kindly provided by Johannes Loffing, University of Zurich, Zurich. Other antibodies include anti-KLHL3 (19 (link)), anti-FLAG, anti-tubulin (all from Sigma), anti-WNK4 (created in the laboratory) (35 (link)), anti-phospho SPAK/OSR1, anti-NCC (all from Millipore), anti-total SPAK, anti-ubiquitin, anti-WNK1 (55 (link)), and anti-calcineurin A (Cell Signaling).

Ishizawa K., Wang Q., Li J., Yamazaki O., Tamura Y., Fujigaki Y., Uchida S., Lifton R.P, & Shibata S. (2019). Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling. Proceedings of the National Academy of Sciences of the United States of America, 116(8), 3155-3160.

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