Journal: Cell reports

Article Title: Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.

doi: 10.1016/j.celrep.2013.02.024

Figure Lengend Snippet: Figure 1. KLHL3 Was Coimmunoprecipitated with Cullin3 and WNK4 (A) FLAG-tagged Cullin3 (CUL3) was coimmunoprecipitated with Halo-tagged KLHL3 in HEK293T cells. IB, immunoblot; IP, immunoprecipitation. (B) T7-tagged WNK4, OSR1, SPAK, and NCC were coexpressed with Halo-tagged KLHL3 in HEK293T cells and immunoprecipitated with T7 antibody. KLHL3 was coimmunoprecipitated only with WNK4. The asterisks indicate T7-tagged proteins. (C) WNK4 was coimmunoprecipitated with endogenous Cullin3 in HEK293T cells in the presence of KLHL3 coexpression. Results similar to those shown in (A), (B), and (C) were obtained in three separate experiments.

Article Snippet: Blots were probed with the following primary antibodies: anti-total NCC (Ohno et al., 2011), anti-phosphorylated NCC (pSer71) (Yang et al., 2007), anti-WNK4 (Ohno et al., 2011; Ohta et al., 2009), anti-total OSR1 (M9; Abnova), anti-phosphorylated OSR1 (Ohta et al., 2009), anti-total SPAK (Cell Signaling Technology), anti-phosphorylated SPAK (Yang et al., 2010), anti-GAPDH (Santa Cruz Biotechnology), anti-actin (Cytoskeleton), anti-HA (Merck Millipore), anti-KLHL3 (Abcam), anti-Cullin3 (Abcam), anti-FLAG (Sigma-Aldrich), anti-Halo (Promega), and anti-T7 (Merck Millipore).

Techniques: Western Blot, Immunoprecipitation

Journal: Cell reports

Article Title: Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.

doi: 10.1016/j.celrep.2013.02.024

Figure Lengend Snippet: Figure 2. Effect of Wild-Type and PHAII-Causing Mutant KLHL3 and Cullin3 Expression on Cellular WNK4 Abundance (A) When coexpressed with KLHL3, the abundance of WNK4 protein within HEK293T cells expressed by CMV promoter (p33FLAG-CMV-10 vector, Sigma- Aldrich) was dramatically decreased, whereas the abundance of BAP expressed by the same expression vector was not affected by KLHL3 coexpression. (B) The reduction of WNK4 by KLHL3 coexpression was confirmed in another expression system of WNK4 (pRK5 vector), and OSR1, SPAK, and NCC levels were not affected by KLHL3 coexpression. Results similar to those shown in (A) and (B) were obtained in three separate experiments. (C) Effect of wild-type and a PHAII-causing mutant (R528H) KLHL3 expression on the cellular abundance of WNK4. Endogenous WNK4 level in mpkDCT cells was decreased by wild-type KLHL3 expression. However, a similar level of the mutant KLHL3 expression failed to reduce WNK4 (*p < 0.05, compared with the WNK4 levels without KLHL3 coexpression [left lane] and with the mutant KLHL3 coexpression [right lane]; n = 3; mean ± SEM). (D) Effect of the endogenous KLHL3 knockdown in mpkDCT cells on WNK4 expression. The protein levels of WNK4 were higher in KLHL3-knocked-down cells than in control cells. siRNA, small interfering RNA. (E) Effect of wild-type and a PHAII-causing mutant Cullin3 expression on the cellular abundance of WNK4. Although the expression of Cullin3 alone did not affect WNK4 protein level, Cullin3 expression with KLHL3 dramatically reduced WNK4 protein. The mutant Cullin3 lacking the portion corresponding to exon 9 was less able to reduce WNK4 protein. The existence of two bands in the immunoblot of Cullin3 was reported previously (McEvoy et al., 2007). Similar results were obtained in three separate experiments.

Article Snippet: Blots were probed with the following primary antibodies: anti-total NCC (Ohno et al., 2011), anti-phosphorylated NCC (pSer71) (Yang et al., 2007), anti-WNK4 (Ohno et al., 2011; Ohta et al., 2009), anti-total OSR1 (M9; Abnova), anti-phosphorylated OSR1 (Ohta et al., 2009), anti-total SPAK (Cell Signaling Technology), anti-phosphorylated SPAK (Yang et al., 2010), anti-GAPDH (Santa Cruz Biotechnology), anti-actin (Cytoskeleton), anti-HA (Merck Millipore), anti-KLHL3 (Abcam), anti-Cullin3 (Abcam), anti-FLAG (Sigma-Aldrich), anti-Halo (Promega), and anti-T7 (Merck Millipore).

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Knockdown, Control, Small Interfering RNA, Western Blot

Journal: Cell reports

Article Title: Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.

doi: 10.1016/j.celrep.2013.02.024

Figure Lengend Snippet: Figure 6. Generation and Analysis of WNK4 TG Mice (A) Status of the WNK-OSR1/SPAK-NCC phosphorylation cascade in the WNK4 TG mice (Tg(Wnk4WT)). WNK4 protein levels in the kidneys of LC (two copies) and HC (thirty copies) TG mice were increased 1.7 ± 0.1- and 9.1 ± 0.2- fold, respectively (*p < 0.05, n = 5, mean ± SEM). NCC phosphorylation in these mice was significantly increased, compared with that of wild-type mice (p < 0.05, n = 5, mean ± SEM). The phosphorylation of SPAK in LC-TG and HC- TG mice and that of OSR1 in HC-TG mice was also significantly increased compared to that of wild-type mice (p < 0.05, n = 5, mean ± SEM). The total and phosphorylated OSR1, SPAK, and NCC in HC-TG mice were significantly increased compared with those in LC-TG mice (p < 0.05, n = 5, mean ± SEM). (B) Nighttime systolic, diastolic, and mean blood pressure (BP) and daytime diastolic blood pressure were significantly increased as the WNK4 protein levels increased in the Tg(Wnk4WT) mice. Values were collected over 7 consecutive days (daytime: from 8:00 until 20:00; nighttime: from 20:00 until 8:00). *p < 0.05 versus WT; yp < 0.05 versus LC; WT n = 5; LC-TG n = 5; HC-TG n = 4; mean ± SEM. See also Figures S3 and S4.

Article Snippet: Blots were probed with the following primary antibodies: anti-total NCC (Ohno et al., 2011), anti-phosphorylated NCC (pSer71) (Yang et al., 2007), anti-WNK4 (Ohno et al., 2011; Ohta et al., 2009), anti-total OSR1 (M9; Abnova), anti-phosphorylated OSR1 (Ohta et al., 2009), anti-total SPAK (Cell Signaling Technology), anti-phosphorylated SPAK (Yang et al., 2010), anti-GAPDH (Santa Cruz Biotechnology), anti-actin (Cytoskeleton), anti-HA (Merck Millipore), anti-KLHL3 (Abcam), anti-Cullin3 (Abcam), anti-FLAG (Sigma-Aldrich), anti-Halo (Promega), and anti-T7 (Merck Millipore).

Techniques: Phospho-proteomics