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1.
FIGURE 8.

FIGURE 8. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

NKCC1 activity in DRG neurons isolated from wild-type and SPAK knockout mice. NKCC1 function was assayed in single DRG cells through bumetanide-sensitive Tl+ uptake. A, individual curves; B, quantitation was performed by averaging the slopes of fluorescent increases calculated using the last baseline data point before addition of TI+ and the first six data points after the addition of Tl+ to the medium. Bars represent mean ± S.E. with six data points for wild-type and three data points for SPAK knockout DRG neurons, respectively.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
2.
FIGURE 2.

FIGURE 2. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

Expression patterns of cotransporters and regulatory kinases. A, expression of NKCC1, KCC1, KCC3, KCC4, OSR1, SPAK, WNK1, WNK2, WNK4, in 50B11 cells, and in rat and mouse DRG neurons were demonstrated by RT-PCR. Absence of WNK3 in 50B11 cells and in rat DRG neurons, compared with mouse DRG neurons is due to the lack of rat-specific WNK3 sequence information. B, central nervous system-specific KCC2 was absent in mouse and rat DRG neurons, as well as in the 50B11 cells, but was present in our positive control brain sample.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
3.
FIGURE 6.

FIGURE 6. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

Effect of SPAK plus OSR1 knockdown on NKCC1 function. A, Western blot analysis showing decreased levels of kinase expression in the different double knockdown (d-KD) clones. B, quantitation based on signals from panel A, corrected for the antibody calibration. C, NKCC1 activity (bumetanide-sensitive K+ flux) was plotted as a function of kinase expression. Data are from wild-type B5011 cells (100% SPAK plus OSR1 expression) and from different 50B11 clones co-transfected with vectors carrying shRNA targeting both SPAK and OSR1. Data were best fitted with a non-linear regression using GraphPad Prism, version 3. Note the close relationship between kinase abundance and NKCC1 activity.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
4.
FIGURE 4.

FIGURE 4. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

Knockdown of OSR-1 and SPAK in 50B11 cells. A, Western blot analysis of OSR1-KD and wild-type 50B11 cells. An equal amount of total protein (250 μg) was loaded per lane. Anti-SPAK and anti-OSR1 were applied individually. Densitometry analysis revealed that OSR-1 expression in OSR1-KD cells was reduced by 65% (n = 3), whereas SPAK expression remained unaltered (not shown). B, K+ fluxes measured through unidirectional 86Rb uptakes demonstrate that NKCC1 activity (bumetanide-sensitive portion of the flux) was significantly reduced (40%) in OSR1-KD cells. C, Western blot analysis of two SPAK-KD clones and wild-type 50B11 cells. An equal amount of total protein (250 μg) was loaded per lane. Anti-SPAK and anti-OSR1 were applied individually. Densitometry analysis revealed that SPAK expression in both SPAK-KD clones was reduced by 60% (n = 3), whereas OSR1 expression remained unchanged (not shown). D, K+ fluxes demonstrate that NKCC1 activity was reduced by 45% in SPAK-KD cells. Bars represent means ± S.E. (n = 12).

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
5.
FIGURE 7.

FIGURE 7. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

NKCC1-mediated Tl+ (thallium) uptake in single isolated DRG neurons. DRG neurons were loaded with the thallium-sensitive dye FluxOR. FluxOR fluorescence (approximate fluorescence excitation and emission: 488/525 nm) increases upon addition of 2.8 mm Tl2SO4 to the external medium. A, photographs of DRG neurons showing increase in fluorescence after a 6 min addition of thallium. The two photographs on the left are differential interference contrast (DIC) pictures, whereas the four photographs on the right are fluorescence pictures. B, fluorescence intensity as a function of time. The top trace represents the fluorescence signal average of 26 DRG neurons under regular conditions. The bottom trace represents the fluorescence signal average of 21 DRG neurons incubated in the presence of 20 μm bumetanide. The difference between the two curves represents the bumetanide-sensitive component of the Tl+ uptake, mediated by NKCC1.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
6.
FIGURE 5.

FIGURE 5. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

shRNA control plus rescue experiments via mSPAK and mOSR1 overexpression. A, Western blot analysis of OSR1 expression in wild-type cells, OSR1-KD cells, OSR1-KD cells rescued by SPAK overexpression, SPAK-KD cells, and SPAK-KD cells rescued with OSR1-HA. An equal amount of total protein (300 μg) except for overexpressing cells (80 μg) was loaded per lane. B, Western blot analysis with anti-HA antibody showing expression of SPAK-HA in OSR1-KD cells and OSR-HA in SPAK-KD cells. Note the higher expression level of mSPAK. An equal amount of protein (150 μg) was loaded per lane. C, control with irrelevant shRNA vector transfection shows no effect on NKCC1 activity. D, mOSR1 was overexpressed in sh-SPAK knockdown 50B11 cells, and mSPAK was overexpressed in sh-OSR1 knockdown cells. Note the increased activity upon SPAK overexpression, correlating with the Western data (panel A). Bars in panels C and D represent means ± S.E. All fluxes were measured in triplicate. Five independent clones expressing the irrelevant shRNA molecule were analyzed. N values are indicated inside the bars.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
7.
FIGURE 1.

FIGURE 1. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

Disruption of the SPAK gene. A, structure of the SPAK gene at exon 6, position of the 5′ probe and structure of the DNA fragment containing the mouse tyrosinase gene, a neomycin resistance gene cassette, and a fragment of the 5′ hprt gene. The position of a unique loxP site is indicated. Note that recombination results in the duplication of a region of the gene (MICER, depicted by a gray box). B, Southern blot analysis of embryonic stem (ES) cell genomic DNA digested with XbaI. The 13.3-kb band represents the control gene, and the 8-kb band originates from the mutant gene. The mutant gene is detected in clones 3F3, 1A2, and 5A12 by the 5′ probe. C, Western blot analysis of 60-mg brain protein isolated from a wild-type and a homozygous mouse. The 60-kDa molecular size band represents SPAK kinase.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.
8.
FIGURE 3.

FIGURE 3. From: The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons.

Calibration of SPAK and OSR1 antibodies and quantitation of SPAK and OSR1 protein abundance in tissues. A, Western blot shows GST-SPAK and GST-OSR1 at two different amounts, probed with anti-GST antibody (1:1000). B, densitometry analysis shows equivalent signals for both full-length proteins (10.2 μl of GST-SPAK elicits the same signal intensity than 10 μl of GST-OSR1). C, quantitation of anti-SPAK and anti-OSR1 antibodies. The same amounts of GST-SPAK and GST-OSR1 were loaded and run in a gel at three different concentrations, transferred, and immunoblotted with anti-SPAK (1:100) and anti-OSR1 (1:1500) antibodies, respectively. D, densitometry analysis shows that anti-SPAK at 1:100 dilution yields a signal 1.7 times stronger than anti-OSR1 at 1:1500 dilution. E and F, Western blot analysis of SPAK and OSR1 expression in DRG, spinal cord, brain cortex, heart, kidney, liver, and 50B11 cells. Equal amount of total protein (200 μg) was loaded per lane. Primary antibodies were used at dilutions of 1:100 for SPAK and 1:1500 for OSR1 to quantitate the level of expression. Densitometry analysis demonstrates that OSR1 and SPAK have similar expression levels in DRG, spinal cord, and liver. Note that, in brain and kidney, there is very little OSR1 expression, whereas in heart, OSR1 expression was twice that of SPAK. SPAK expression in 50B11 cells was shown to be 1.5 times higher than OSR1 expression.

Yang Geng, et al. J Biol Chem. 2009 May 22;284(21):14020-14028.

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