Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Confirmation of the in vitro phosphorylation reaction of GST-NKCC2 using three different anti-phospho-NKCC2 antibodies. GST-NKCC2 is incubated with GST-SPAK [T233E] in the absence or presence of MO25α. Subsequently, the GST-NKCC2 phosphorylation reaction is confirmed by the immunoblotting by using three different anti-phospho-NKCC2 antibodies: anti-pNKCC2 (pT2), pNKCC2 (pThr100), and pNKCC2 (pThr100/105)[pNCC (pThr50/55)]. The most dramatic increase in ATP-dependent NKCC2 phosphorylation is observed in the presence of GST-MO25α.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: In Vitro, Phospho-proteomics, Incubation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Kinase assay and detection of phosphorylated NKCC2 in the ELISA system. GST-NKCC2 [1–174] is coated to each well of the ELISA plate and incubated with GST-SPAK [T233E] in the presence of GST-MO25α. Anti-p-NKCC2 (pT2) and p-NKCC2 (pThr100/105) [p-NCC (pTh50/55)] antibodies succeed in detecting NKCC2 phosphorylation. Anti-p-NKCC2 (pThr100) antibody is not appropriate for ELISA, because a nonspecific blue color change also develops in negative control wells.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Kinase Assay, Enzyme-linked Immunosorbent Assay, Incubation, Phospho-proteomics, Negative Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Representative figure of primary screening of SPAK inhibitors in the newly developed ELISA system. Each compound is added in the kinase reaction at a final concentration of 50 μM. As a positive control indicating nonphosphorylation of NKCC2, ATP-free kinase buffer is used. A kinase reaction without any compounds is used as a negative control for screening. The arbitrary cut-off value of OD620 is set to 0.2. The compound shown as a white bar is regarded as a positive compound having a strong inhibitory effect on SPAK-regulated NKCC2 phosphorylation.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Positive Control, Negative Control, Phospho-proteomics

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Inhibitory effect of 1S-14279 on WNK-SPAK-NCC/NKCC1 signaling in mouse kidney and aorta. (A) Representative immunoblots of total- and p-NCC, NKCC2, and p38 of the kidney at 30 minutes after infusion of 1S-14279. The NCC phosphorylation is drastically reduced by 1S-14279. No significant increases in pNKCC2 and p-p38 levels are observed. The lower panel shows quantification of the results of the blots (n=4, mean±SEM). (B) Representative immunoblots of total- and p-NKCC1 of the aorta at 30 minutes after infusion of 1S-14279. The expression of p-NKCC1 is markedly reduced. No significant difference in total NKCC1 abundance. The lower panel shows quantification of the results of the blots (n=4, mean±SEM). *P<0.05; **P<0.01. Ctrl, control.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Western Blot, Phospho-proteomics, Expressing, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Inhibitory effect of Closantel on WNK-SPAK-NCC/NKCC1 signaling in mouse kidney and aorta. (A) Representative immunoblots of total- and p-NCC, NKCC2 and p38 of the kidney at 30 minutes after infusion of Closantel. The NCC phosphorylation is drastically reduced by Closantel. No significant increases in pNKCC2 and p-p38 levels are observed. The lower panel shows quantification of the results of the blots (n=3, mean±SEM). (B) Representative immunoblots of total- and p-NKCC1 of the aorta at 30 minutes after infusion of Closantel. The expression of p-NKCC1 is markedly reduced. No significant difference in total NKCC1 abundance. The lower panel shows quantification of the results of the blots (n=4, mean±SEM). *P<0.05; **P<0.01. Ctrl, control.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Western Blot, Phospho-proteomics, Expressing, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Inhibitory effect of chronic Closantel treatment on WNK-SPAK-NCC/NKCC1 signaling in mouse kidney and aorta. The dosage of Closantel added to the mouse chow is 300 mg/kg per day. (A) Representative immunoblots of total- and p-NCC and NKCC2 after 7 days treatment of Closantel. NCC and NKCC2 phosphorylation are markedly reduced by Closantel. The lower panel shows the quantification of the results of the blots (n=4, mean±SEM). (B) Representative immunoblots for the total- and p-NKCC1 of the aorta after 7 days Closantel administration. The expression of p-NKCC1 is markedly reduced. The lower panel shows the quantification of the results of the blots (n=4, mean±SEM). *P<0.05; **P<0.01.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Western Blot, Phospho-proteomics, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Inhibitory effect of 1S-14279 on WNK-SPAK-NCC/NKCC1 signaling in mouse kidney and aorta. (A) Representative immunoblots of total- and p-NCC, NKCC2, and p38 of the kidney at 30 minutes after infusion of 1S-14279. The NCC phosphorylation is drastically reduced by 1S-14279. No significant increases in pNKCC2 and p-p38 levels are observed. The lower panel shows quantification of the results of the blots (n=4, mean±SEM). (B) Representative immunoblots of total- and p-NKCC1 of the aorta at 30 minutes after infusion of 1S-14279. The expression of p-NKCC1 is markedly reduced. No significant difference in total NKCC1 abundance. The lower panel shows quantification of the results of the blots (n=4, mean±SEM). *P<0.05; **P<0.01. Ctrl, control.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Western Blot, Phospho-proteomics, Expressing, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

doi: 10.1681/ASN.2014060560

Figure Lengend Snippet: Inhibitory effect of Closantel on WNK-SPAK-NCC/NKCC1 signaling in mouse kidney and aorta. (A) Representative immunoblots of total- and p-NCC, NKCC2 and p38 of the kidney at 30 minutes after infusion of Closantel. The NCC phosphorylation is drastically reduced by Closantel. No significant increases in pNKCC2 and p-p38 levels are observed. The lower panel shows quantification of the results of the blots (n=3, mean±SEM). (B) Representative immunoblots of total- and p-NKCC1 of the aorta at 30 minutes after infusion of Closantel. The expression of p-NKCC1 is markedly reduced. No significant difference in total NKCC1 abundance. The lower panel shows quantification of the results of the blots (n=4, mean±SEM). *P<0.05; **P<0.01. Ctrl, control.

Article Snippet: 4 Blots were probed with the following primary antibodies: anti-total NCC, 35 anti-phosphorylated NCC (pThr53 and pSer71 in mouse NCC), 11 anti-total NKCC1 (T4) (Hybridoma Bank, University of Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1), 27 anti-total NKCC2 (Alpha Diagnostic, San Antonio, TX), anti-phosphorylated NKCC2 (pThr100 in human NKCC2), 36 anti-phosphorylated NKCC2 (termed pT2 antibody; kindly provided by K. Mutig), 37 anti-phosphorylated p38, anti-total p38 (Cell Signaling Technology, Danvers, MA), and anti-actin (Cytoskeleton, Denver, CO).

Techniques: Western Blot, Phospho-proteomics, Expressing, Control