romk  (Alomone Labs)


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    Structured Review

    Alomone Labs romk
    3D perception of the <t>GFP-labeled</t> <t>ROMK</t> channels in a representative adult mouse heart following gene therapy. The 3D physical presences of ( A ) epicardium (red) and ( B ) ventricular cavity (yellow) are superimposed on the ( C ) animated heart. ( D ) 3D distribution of ROMK channels (green) is superimposed with C to provide a ( E ) physiological model. ( F ) The distribution of ROMK channels in the atrium. ( G ) The exploration of the animated ventricular cavity or ROMK channels. ( H ) This illustration integrates the epicardium (red), ventricular cavity (yellow), and ROMK channels (green) for an interactive and immersive experience. Scale bar: 1 mm. ( I – K ) VR-LSFM allows for reading instructive texts by clicking the 3D anatomic features of ( I ) the atrium, ( J ) the ventricular cavity, and ( K ) ROMK channels. ( L–N ) VR-LSFM enables zooming into the 3D anatomy to interrogate ROMK channels in relation to the ventricular cavity and myocardium. ROMK, renal outer medullary potassium; VR, virtual reality; LSFM, light-sheet fluorescence microscopy. All of these images are shown in pseudocolor.
    Romk, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/romk/product/Alomone Labs
    Average 93 stars, based on 3 article reviews
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    romk - by Bioz Stars, 2022-11
    93/100 stars

    Images

    1) Product Images from "Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics"

    Article Title: Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics

    Journal: JCI Insight

    doi: 10.1172/jci.insight.97180

    3D perception of the GFP-labeled ROMK channels in a representative adult mouse heart following gene therapy. The 3D physical presences of ( A ) epicardium (red) and ( B ) ventricular cavity (yellow) are superimposed on the ( C ) animated heart. ( D ) 3D distribution of ROMK channels (green) is superimposed with C to provide a ( E ) physiological model. ( F ) The distribution of ROMK channels in the atrium. ( G ) The exploration of the animated ventricular cavity or ROMK channels. ( H ) This illustration integrates the epicardium (red), ventricular cavity (yellow), and ROMK channels (green) for an interactive and immersive experience. Scale bar: 1 mm. ( I – K ) VR-LSFM allows for reading instructive texts by clicking the 3D anatomic features of ( I ) the atrium, ( J ) the ventricular cavity, and ( K ) ROMK channels. ( L–N ) VR-LSFM enables zooming into the 3D anatomy to interrogate ROMK channels in relation to the ventricular cavity and myocardium. ROMK, renal outer medullary potassium; VR, virtual reality; LSFM, light-sheet fluorescence microscopy. All of these images are shown in pseudocolor.
    Figure Legend Snippet: 3D perception of the GFP-labeled ROMK channels in a representative adult mouse heart following gene therapy. The 3D physical presences of ( A ) epicardium (red) and ( B ) ventricular cavity (yellow) are superimposed on the ( C ) animated heart. ( D ) 3D distribution of ROMK channels (green) is superimposed with C to provide a ( E ) physiological model. ( F ) The distribution of ROMK channels in the atrium. ( G ) The exploration of the animated ventricular cavity or ROMK channels. ( H ) This illustration integrates the epicardium (red), ventricular cavity (yellow), and ROMK channels (green) for an interactive and immersive experience. Scale bar: 1 mm. ( I – K ) VR-LSFM allows for reading instructive texts by clicking the 3D anatomic features of ( I ) the atrium, ( J ) the ventricular cavity, and ( K ) ROMK channels. ( L–N ) VR-LSFM enables zooming into the 3D anatomy to interrogate ROMK channels in relation to the ventricular cavity and myocardium. ROMK, renal outer medullary potassium; VR, virtual reality; LSFM, light-sheet fluorescence microscopy. All of these images are shown in pseudocolor.

    Techniques Used: Labeling, Fluorescence, Microscopy

    2) Product Images from "Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels"

    Article Title: Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013121326

    Lovastatin stimulates ROMK1 channels through a cholesterol-associated, PI(4)5K I γ -dependent mechanism. (A) Representative single-channel records from cell-attached patches formed on a control cell, a cell treated for 12 hours with 10 μ M lovastatin alone, a cell treated for 12 hours with 10 μ g/ml cholesterol alone, or a cell treated for 12 hours with 10 μ M lovastatin plus 10 μ g/ml cholesterol when these cells were either (left panel) under control conditions or (right panel) transfected with siRNA of PI(4)P5K I γ . (B) Summary plots of ROMK1 P O under these conditions. (C) Representative Western blot from four separate experiments consistently shows that transfection of mpkCCD c14 cells with siRNA against PI(4)5K I γ reduced PI(4)5K I γ expression.
    Figure Legend Snippet: Lovastatin stimulates ROMK1 channels through a cholesterol-associated, PI(4)5K I γ -dependent mechanism. (A) Representative single-channel records from cell-attached patches formed on a control cell, a cell treated for 12 hours with 10 μ M lovastatin alone, a cell treated for 12 hours with 10 μ g/ml cholesterol alone, or a cell treated for 12 hours with 10 μ M lovastatin plus 10 μ g/ml cholesterol when these cells were either (left panel) under control conditions or (right panel) transfected with siRNA of PI(4)P5K I γ . (B) Summary plots of ROMK1 P O under these conditions. (C) Representative Western blot from four separate experiments consistently shows that transfection of mpkCCD c14 cells with siRNA against PI(4)5K I γ reduced PI(4)5K I γ expression.

    Techniques Used: Transfection, Western Blot, Expressing

    ROMK1 channel is expressed in the apical membrane of mpkCCD c14 cells. (A, Left panel) Current-voltage relationship summarized from six cell-attached patches. (A, Right panel) Single-channel currents from a representative cell-attached patch versus the potentials applied to the patch pipette ( V pipette ), which indicated by − V pipette (millivolts). C- and dashed lines along with single-channel current traces show baselines where the channels are closed. (B) Channel activity in (left panel) an inside-out patch was blocked by 10 mM tetraethylammonium (TEA) applied to (center panel) the cytoplasmic bath, and (right panel) the blockade was reversed after washing TEA out of the bath. A voltage step protocol from −80 to 80 mV with 80-mV increments at a holding potential of 0 mV, as indicated by V m (millivolts), was applied to the patch pipette. (C) Appearance of the channel in the patch was significantly reduced in the cells transfected with ROMK1 siRNA but not control siRNA. Downward events show inward currents through open channels. (D) Summary plots of percentages of patches containing ROMK1 channel activity in cell-attached patches formed on control cells ( n =20) or cells transfected with either ROMK1 siRNA ( n =18) or control siRNA ( n =16). Data are from four separate experiments. (E) Western blot from control cells or cells transfected with either ROMK1 siRNA or control siRNA. β -actin in the same loading membrane was immunoprecipitated with antibody to β -actin and used to evaluate the amount of loaded protein. (F) Summary plots of relative ROMK1 protein levels from four separate Western blot experiments.
    Figure Legend Snippet: ROMK1 channel is expressed in the apical membrane of mpkCCD c14 cells. (A, Left panel) Current-voltage relationship summarized from six cell-attached patches. (A, Right panel) Single-channel currents from a representative cell-attached patch versus the potentials applied to the patch pipette ( V pipette ), which indicated by − V pipette (millivolts). C- and dashed lines along with single-channel current traces show baselines where the channels are closed. (B) Channel activity in (left panel) an inside-out patch was blocked by 10 mM tetraethylammonium (TEA) applied to (center panel) the cytoplasmic bath, and (right panel) the blockade was reversed after washing TEA out of the bath. A voltage step protocol from −80 to 80 mV with 80-mV increments at a holding potential of 0 mV, as indicated by V m (millivolts), was applied to the patch pipette. (C) Appearance of the channel in the patch was significantly reduced in the cells transfected with ROMK1 siRNA but not control siRNA. Downward events show inward currents through open channels. (D) Summary plots of percentages of patches containing ROMK1 channel activity in cell-attached patches formed on control cells ( n =20) or cells transfected with either ROMK1 siRNA ( n =18) or control siRNA ( n =16). Data are from four separate experiments. (E) Western blot from control cells or cells transfected with either ROMK1 siRNA or control siRNA. β -actin in the same loading membrane was immunoprecipitated with antibody to β -actin and used to evaluate the amount of loaded protein. (F) Summary plots of relative ROMK1 protein levels from four separate Western blot experiments.

    Techniques Used: Transferring, Activity Assay, Transfection, Western Blot, Immunoprecipitation

    A major fraction of ROMK1 channels is located in planar regions. (A and B) Atomic force microscopy shows that acute extraction of cholesterol with M β CD (50 mM for 30 minutes) but not lovastatin (10 μ M for 12 hours) eliminated microvilli (A, representative image; B, summary plots of microvillar height). (C) Depiction of the methods used for preparation of the whole apical membrane and separation of microvilli from planar regions. Biotinylation of either the whole apical membrane or the remaining membrane after washing out M β CD-released vesicles (planar regions) was used for lane 1 or 2, respectively, in the Western blots shown in D, whereas M β CD-released vesicles (microvilli) were used for lane 3. M β CD excised microvilli to form vesicles, which are indicated by red arrows. The white rectangular box in C indicates the zoomed-in area shown in the Zoom-in panel. (D) Western blots of the whole apical membrane (whole membrane; lane 1), the remaining apical membrane after washing out vesicles (planar regions; lane 2), and M β . (E) Summary plots of relative ROMK1/prominin-1 distribution between planar regions and microvilli ( n . First, we immunoblotted the membrane with an antibody to ROMK1. Second, the lower part of the membrane that carries prominin-1 was separated from the top part of the membrane that carries ROMK1 and immunoblotted with an antibody to prominin-1. The arrows in C indicate where the separated membrane was fused back together to show that they were from the same gel. In the same lane for microvilli, where ROMK1 was undetectable, significant prominin-1 was observed. Conversely, in the same lane for planar regions, where prominin-1 was undetectable, significant ROMK1 was observed. These data suggest that neither undetectable ROMK1 in microvilli nor undetectable prominin-1 in planar regions are caused by an insufficient protein loading.
    Figure Legend Snippet: A major fraction of ROMK1 channels is located in planar regions. (A and B) Atomic force microscopy shows that acute extraction of cholesterol with M β CD (50 mM for 30 minutes) but not lovastatin (10 μ M for 12 hours) eliminated microvilli (A, representative image; B, summary plots of microvillar height). (C) Depiction of the methods used for preparation of the whole apical membrane and separation of microvilli from planar regions. Biotinylation of either the whole apical membrane or the remaining membrane after washing out M β CD-released vesicles (planar regions) was used for lane 1 or 2, respectively, in the Western blots shown in D, whereas M β CD-released vesicles (microvilli) were used for lane 3. M β CD excised microvilli to form vesicles, which are indicated by red arrows. The white rectangular box in C indicates the zoomed-in area shown in the Zoom-in panel. (D) Western blots of the whole apical membrane (whole membrane; lane 1), the remaining apical membrane after washing out vesicles (planar regions; lane 2), and M β . (E) Summary plots of relative ROMK1/prominin-1 distribution between planar regions and microvilli ( n . First, we immunoblotted the membrane with an antibody to ROMK1. Second, the lower part of the membrane that carries prominin-1 was separated from the top part of the membrane that carries ROMK1 and immunoblotted with an antibody to prominin-1. The arrows in C indicate where the separated membrane was fused back together to show that they were from the same gel. In the same lane for microvilli, where ROMK1 was undetectable, significant prominin-1 was observed. Conversely, in the same lane for planar regions, where prominin-1 was undetectable, significant ROMK1 was observed. These data suggest that neither undetectable ROMK1 in microvilli nor undetectable prominin-1 in planar regions are caused by an insufficient protein loading.

    Techniques Used: Microscopy, Western Blot

    ROMK1 channels are not located in lipid rafts. (A) Sucrose gradient experiments showed that ROMK1 is located in nonlipid raft regions. Caveolin-1 (Cav-1) was used as a control protein that is known to be located in lipid rafts, whereas Rab5 was used as a control protein that is known to be located in nonlipid raft membranes. IB, immunoblotting. (B) Summary plots of four sucrose gradient experiments. (C) Confocal microscopy fluorescent image merged with a DIC image shows that CTX (green) is mainly located in microvilli (microvilli were visualized through DIC imaging). (D) Confocal microscopy shows that ROMK1 (red) is not located in lipid rafts probed by fluorescence-tagged CTX (green). Here and in other figures, all confocal microscopy xy optical sections were taken near the apical membrane of mpkCCD c14 cells, which is evidenced by tight junctions (white arrows); white rectangular boxes indicate zoomed-in areas shown in the Zoom-in panels. Frequency of pixels was analyzed with the ImageJ program, and the images represent data from at least four separate experiments that showed consistent results. All confocal microscopy images in this study were taken using the same parameter settings, including gain, contrast, and pinhole. DIC, differential interference contrast.
    Figure Legend Snippet: ROMK1 channels are not located in lipid rafts. (A) Sucrose gradient experiments showed that ROMK1 is located in nonlipid raft regions. Caveolin-1 (Cav-1) was used as a control protein that is known to be located in lipid rafts, whereas Rab5 was used as a control protein that is known to be located in nonlipid raft membranes. IB, immunoblotting. (B) Summary plots of four sucrose gradient experiments. (C) Confocal microscopy fluorescent image merged with a DIC image shows that CTX (green) is mainly located in microvilli (microvilli were visualized through DIC imaging). (D) Confocal microscopy shows that ROMK1 (red) is not located in lipid rafts probed by fluorescence-tagged CTX (green). Here and in other figures, all confocal microscopy xy optical sections were taken near the apical membrane of mpkCCD c14 cells, which is evidenced by tight junctions (white arrows); white rectangular boxes indicate zoomed-in areas shown in the Zoom-in panels. Frequency of pixels was analyzed with the ImageJ program, and the images represent data from at least four separate experiments that showed consistent results. All confocal microscopy images in this study were taken using the same parameter settings, including gain, contrast, and pinhole. DIC, differential interference contrast.

    Techniques Used: Confocal Microscopy, Imaging, Fluorescence

    ROMK1 channels are mainly located in the planar region. (A–C) Confocal microscopy images of prominin-1 (a biomarker of microvilli; green) merged with ROMK1 (red) show that ROMK1 was separated from prominin-1 in control mpkCCD c14 cells and that ROMK1 was almost undetectable in mpkCCD c14 cells treated with ROMK1 siRNA but still observed in mpkCCD c14 cells treated with control siRNA. (D) Fluorescent intensity of either prominin-1 (green) or ROMK1 (red) was quantified with the ImageJ program, showing that ROMK1 levels were significantly reduced in ROMK1 siRNA-treated cells.
    Figure Legend Snippet: ROMK1 channels are mainly located in the planar region. (A–C) Confocal microscopy images of prominin-1 (a biomarker of microvilli; green) merged with ROMK1 (red) show that ROMK1 was separated from prominin-1 in control mpkCCD c14 cells and that ROMK1 was almost undetectable in mpkCCD c14 cells treated with ROMK1 siRNA but still observed in mpkCCD c14 cells treated with control siRNA. (D) Fluorescent intensity of either prominin-1 (green) or ROMK1 (red) was quantified with the ImageJ program, showing that ROMK1 levels were significantly reduced in ROMK1 siRNA-treated cells.

    Techniques Used: Confocal Microscopy, Biomarker Assay

    ROMK1 on Western blots runs at a higher molecular mass than expected in cultured mpkCCDc14 cells. (A) Western blots from mpkCCD c14 cells or kidney tissue of wild-type (WT) or ROMK knockout (KO) mice. (Left panel) ROMK1 channels in the same gel were first probed with a commercial ROMK1 antibody from Alomone Laboratories (Alomone Ab), and then, the membrane was completely stripped and reprobed with another ROMK1 antibody provided by Paul Welling at the University of Maryland Medical School (R79 AP-4 Ab). (Right panel) ROMK1 channels in a different gel were also detected with R79 AP-4 Ab. (B) Western blot from either control mpkCCD c14 cells or the cells treated for 24 hours with 250 μ M TEMPOL, a superoxide dismutase mimetic and ROS scavenger. (C) Both the higher molecular mass form of ROMK1 and the lower molecular mass form were detected in lysate from whole kidney, whereas only the higher molecular mass form of ROMK1 was detected in apical membrane protein extract from in situ biotinylated renal tubules. (D) Treatment of apical membrane protein extract of renal tubules with 100 mM DTT decreased the higher molecular mass form and increased the lower molecular mass form of ROMK1. ( E ) Summary plots of the two forms of ROMK1 in apical membranes of renal tubules in the absence or presence of DTT. Methods for preparing cell lysates are the same for mpkCCD c14 cells and kidney tissue. Each experiment was repeated three times and showed consistent results. DTT, dithiothreitol.
    Figure Legend Snippet: ROMK1 on Western blots runs at a higher molecular mass than expected in cultured mpkCCDc14 cells. (A) Western blots from mpkCCD c14 cells or kidney tissue of wild-type (WT) or ROMK knockout (KO) mice. (Left panel) ROMK1 channels in the same gel were first probed with a commercial ROMK1 antibody from Alomone Laboratories (Alomone Ab), and then, the membrane was completely stripped and reprobed with another ROMK1 antibody provided by Paul Welling at the University of Maryland Medical School (R79 AP-4 Ab). (Right panel) ROMK1 channels in a different gel were also detected with R79 AP-4 Ab. (B) Western blot from either control mpkCCD c14 cells or the cells treated for 24 hours with 250 μ M TEMPOL, a superoxide dismutase mimetic and ROS scavenger. (C) Both the higher molecular mass form of ROMK1 and the lower molecular mass form were detected in lysate from whole kidney, whereas only the higher molecular mass form of ROMK1 was detected in apical membrane protein extract from in situ biotinylated renal tubules. (D) Treatment of apical membrane protein extract of renal tubules with 100 mM DTT decreased the higher molecular mass form and increased the lower molecular mass form of ROMK1. ( E ) Summary plots of the two forms of ROMK1 in apical membranes of renal tubules in the absence or presence of DTT. Methods for preparing cell lysates are the same for mpkCCD c14 cells and kidney tissue. Each experiment was repeated three times and showed consistent results. DTT, dithiothreitol.

    Techniques Used: Western Blot, Cell Culture, Knock-Out, Mouse Assay, In Situ

    3) Product Images from "Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels"

    Article Title: Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013121326

    Lovastatin stimulates ROMK1 channels through a cholesterol-associated, PI(4)5K I γ -dependent mechanism. (A) Representative single-channel records from cell-attached patches formed on a control cell, a cell treated for 12 hours with 10 μ M lovastatin alone, a cell treated for 12 hours with 10 μ g/ml cholesterol alone, or a cell treated for 12 hours with 10 μ M lovastatin plus 10 μ g/ml cholesterol when these cells were either (left panel) under control conditions or (right panel) transfected with siRNA of PI(4)P5K I γ . (B) Summary plots of ROMK1 P O under these conditions. (C) Representative Western blot from four separate experiments consistently shows that transfection of mpkCCD c14 cells with siRNA against PI(4)5K I γ reduced PI(4)5K I γ expression.
    Figure Legend Snippet: Lovastatin stimulates ROMK1 channels through a cholesterol-associated, PI(4)5K I γ -dependent mechanism. (A) Representative single-channel records from cell-attached patches formed on a control cell, a cell treated for 12 hours with 10 μ M lovastatin alone, a cell treated for 12 hours with 10 μ g/ml cholesterol alone, or a cell treated for 12 hours with 10 μ M lovastatin plus 10 μ g/ml cholesterol when these cells were either (left panel) under control conditions or (right panel) transfected with siRNA of PI(4)P5K I γ . (B) Summary plots of ROMK1 P O under these conditions. (C) Representative Western blot from four separate experiments consistently shows that transfection of mpkCCD c14 cells with siRNA against PI(4)5K I γ reduced PI(4)5K I γ expression.

    Techniques Used: Transfection, Western Blot, Expressing

    ROMK1 channel is expressed in the apical membrane of mpkCCD c14 cells. (A, Left panel) Current-voltage relationship summarized from six cell-attached patches. (A, Right panel) Single-channel currents from a representative cell-attached patch versus the potentials applied to the patch pipette ( V pipette ), which indicated by − V pipette (millivolts). C- and dashed lines along with single-channel current traces show baselines where the channels are closed. (B) Channel activity in (left panel) an inside-out patch was blocked by 10 mM tetraethylammonium (TEA) applied to (center panel) the cytoplasmic bath, and (right panel) the blockade was reversed after washing TEA out of the bath. A voltage step protocol from −80 to 80 mV with 80-mV increments at a holding potential of 0 mV, as indicated by V m (millivolts), was applied to the patch pipette. (C) Appearance of the channel in the patch was significantly reduced in the cells transfected with ROMK1 siRNA but not control siRNA. Downward events show inward currents through open channels. (D) Summary plots of percentages of patches containing ROMK1 channel activity in cell-attached patches formed on control cells ( n =20) or cells transfected with either ROMK1 siRNA ( n =18) or control siRNA ( n =16). Data are from four separate experiments. (E) Western blot from control cells or cells transfected with either ROMK1 siRNA or control siRNA. β -actin in the same loading membrane was immunoprecipitated with antibody to β -actin and used to evaluate the amount of loaded protein. (F) Summary plots of relative ROMK1 protein levels from four separate Western blot experiments.
    Figure Legend Snippet: ROMK1 channel is expressed in the apical membrane of mpkCCD c14 cells. (A, Left panel) Current-voltage relationship summarized from six cell-attached patches. (A, Right panel) Single-channel currents from a representative cell-attached patch versus the potentials applied to the patch pipette ( V pipette ), which indicated by − V pipette (millivolts). C- and dashed lines along with single-channel current traces show baselines where the channels are closed. (B) Channel activity in (left panel) an inside-out patch was blocked by 10 mM tetraethylammonium (TEA) applied to (center panel) the cytoplasmic bath, and (right panel) the blockade was reversed after washing TEA out of the bath. A voltage step protocol from −80 to 80 mV with 80-mV increments at a holding potential of 0 mV, as indicated by V m (millivolts), was applied to the patch pipette. (C) Appearance of the channel in the patch was significantly reduced in the cells transfected with ROMK1 siRNA but not control siRNA. Downward events show inward currents through open channels. (D) Summary plots of percentages of patches containing ROMK1 channel activity in cell-attached patches formed on control cells ( n =20) or cells transfected with either ROMK1 siRNA ( n =18) or control siRNA ( n =16). Data are from four separate experiments. (E) Western blot from control cells or cells transfected with either ROMK1 siRNA or control siRNA. β -actin in the same loading membrane was immunoprecipitated with antibody to β -actin and used to evaluate the amount of loaded protein. (F) Summary plots of relative ROMK1 protein levels from four separate Western blot experiments.

    Techniques Used: Transferring, Activity Assay, Transfection, Western Blot, Immunoprecipitation

    A major fraction of ROMK1 channels is located in planar regions. (A and B) Atomic force microscopy shows that acute extraction of cholesterol with M β CD (50 mM for 30 minutes) but not lovastatin (10 μ M for 12 hours) eliminated microvilli (A, representative image; B, summary plots of microvillar height). (C) Depiction of the methods used for preparation of the whole apical membrane and separation of microvilli from planar regions. Biotinylation of either the whole apical membrane or the remaining membrane after washing out M β CD-released vesicles (planar regions) was used for lane 1 or 2, respectively, in the Western blots shown in D, whereas M β CD-released vesicles (microvilli) were used for lane 3. M β CD excised microvilli to form vesicles, which are indicated by red arrows. The white rectangular box in C indicates the zoomed-in area shown in the Zoom-in panel. (D) Western blots of the whole apical membrane (whole membrane; lane 1), the remaining apical membrane after washing out vesicles (planar regions; lane 2), and M β . (E) Summary plots of relative ROMK1/prominin-1 distribution between planar regions and microvilli ( n . First, we immunoblotted the membrane with an antibody to ROMK1. Second, the lower part of the membrane that carries prominin-1 was separated from the top part of the membrane that carries ROMK1 and immunoblotted with an antibody to prominin-1. The arrows in C indicate where the separated membrane was fused back together to show that they were from the same gel. In the same lane for microvilli, where ROMK1 was undetectable, significant prominin-1 was observed. Conversely, in the same lane for planar regions, where prominin-1 was undetectable, significant ROMK1 was observed. These data suggest that neither undetectable ROMK1 in microvilli nor undetectable prominin-1 in planar regions are caused by an insufficient protein loading.
    Figure Legend Snippet: A major fraction of ROMK1 channels is located in planar regions. (A and B) Atomic force microscopy shows that acute extraction of cholesterol with M β CD (50 mM for 30 minutes) but not lovastatin (10 μ M for 12 hours) eliminated microvilli (A, representative image; B, summary plots of microvillar height). (C) Depiction of the methods used for preparation of the whole apical membrane and separation of microvilli from planar regions. Biotinylation of either the whole apical membrane or the remaining membrane after washing out M β CD-released vesicles (planar regions) was used for lane 1 or 2, respectively, in the Western blots shown in D, whereas M β CD-released vesicles (microvilli) were used for lane 3. M β CD excised microvilli to form vesicles, which are indicated by red arrows. The white rectangular box in C indicates the zoomed-in area shown in the Zoom-in panel. (D) Western blots of the whole apical membrane (whole membrane; lane 1), the remaining apical membrane after washing out vesicles (planar regions; lane 2), and M β . (E) Summary plots of relative ROMK1/prominin-1 distribution between planar regions and microvilli ( n . First, we immunoblotted the membrane with an antibody to ROMK1. Second, the lower part of the membrane that carries prominin-1 was separated from the top part of the membrane that carries ROMK1 and immunoblotted with an antibody to prominin-1. The arrows in C indicate where the separated membrane was fused back together to show that they were from the same gel. In the same lane for microvilli, where ROMK1 was undetectable, significant prominin-1 was observed. Conversely, in the same lane for planar regions, where prominin-1 was undetectable, significant ROMK1 was observed. These data suggest that neither undetectable ROMK1 in microvilli nor undetectable prominin-1 in planar regions are caused by an insufficient protein loading.

    Techniques Used: Microscopy, Western Blot

    ROMK1 channels are not located in lipid rafts. (A) Sucrose gradient experiments showed that ROMK1 is located in nonlipid raft regions. Caveolin-1 (Cav-1) was used as a control protein that is known to be located in lipid rafts, whereas Rab5 was used as a control protein that is known to be located in nonlipid raft membranes. IB, immunoblotting. (B) Summary plots of four sucrose gradient experiments. (C) Confocal microscopy fluorescent image merged with a DIC image shows that CTX (green) is mainly located in microvilli (microvilli were visualized through DIC imaging). (D) Confocal microscopy shows that ROMK1 (red) is not located in lipid rafts probed by fluorescence-tagged CTX (green). Here and in other figures, all confocal microscopy xy optical sections were taken near the apical membrane of mpkCCD c14 cells, which is evidenced by tight junctions (white arrows); white rectangular boxes indicate zoomed-in areas shown in the Zoom-in panels. Frequency of pixels was analyzed with the ImageJ program, and the images represent data from at least four separate experiments that showed consistent results. All confocal microscopy images in this study were taken using the same parameter settings, including gain, contrast, and pinhole. DIC, differential interference contrast.
    Figure Legend Snippet: ROMK1 channels are not located in lipid rafts. (A) Sucrose gradient experiments showed that ROMK1 is located in nonlipid raft regions. Caveolin-1 (Cav-1) was used as a control protein that is known to be located in lipid rafts, whereas Rab5 was used as a control protein that is known to be located in nonlipid raft membranes. IB, immunoblotting. (B) Summary plots of four sucrose gradient experiments. (C) Confocal microscopy fluorescent image merged with a DIC image shows that CTX (green) is mainly located in microvilli (microvilli were visualized through DIC imaging). (D) Confocal microscopy shows that ROMK1 (red) is not located in lipid rafts probed by fluorescence-tagged CTX (green). Here and in other figures, all confocal microscopy xy optical sections were taken near the apical membrane of mpkCCD c14 cells, which is evidenced by tight junctions (white arrows); white rectangular boxes indicate zoomed-in areas shown in the Zoom-in panels. Frequency of pixels was analyzed with the ImageJ program, and the images represent data from at least four separate experiments that showed consistent results. All confocal microscopy images in this study were taken using the same parameter settings, including gain, contrast, and pinhole. DIC, differential interference contrast.

    Techniques Used: Confocal Microscopy, Imaging, Fluorescence

    ROMK1 channels are mainly located in the planar region. (A–C) Confocal microscopy images of prominin-1 (a biomarker of microvilli; green) merged with ROMK1 (red) show that ROMK1 was separated from prominin-1 in control mpkCCD c14 cells and that ROMK1 was almost undetectable in mpkCCD c14 cells treated with ROMK1 siRNA but still observed in mpkCCD c14 cells treated with control siRNA. (D) Fluorescent intensity of either prominin-1 (green) or ROMK1 (red) was quantified with the ImageJ program, showing that ROMK1 levels were significantly reduced in ROMK1 siRNA-treated cells.
    Figure Legend Snippet: ROMK1 channels are mainly located in the planar region. (A–C) Confocal microscopy images of prominin-1 (a biomarker of microvilli; green) merged with ROMK1 (red) show that ROMK1 was separated from prominin-1 in control mpkCCD c14 cells and that ROMK1 was almost undetectable in mpkCCD c14 cells treated with ROMK1 siRNA but still observed in mpkCCD c14 cells treated with control siRNA. (D) Fluorescent intensity of either prominin-1 (green) or ROMK1 (red) was quantified with the ImageJ program, showing that ROMK1 levels were significantly reduced in ROMK1 siRNA-treated cells.

    Techniques Used: Confocal Microscopy, Biomarker Assay

    ROMK1 on Western blots runs at a higher molecular mass than expected in cultured mpkCCDc14 cells. (A) Western blots from mpkCCD c14 cells or kidney tissue of wild-type (WT) or ROMK knockout (KO) mice. (Left panel) ROMK1 channels in the same gel were first probed with a commercial ROMK1 antibody from Alomone Laboratories (Alomone Ab), and then, the membrane was completely stripped and reprobed with another ROMK1 antibody provided by Paul Welling at the University of Maryland Medical School (R79 AP-4 Ab). (Right panel) ROMK1 channels in a different gel were also detected with R79 AP-4 Ab. (B) Western blot from either control mpkCCD c14 cells or the cells treated for 24 hours with 250 μ M TEMPOL, a superoxide dismutase mimetic and ROS scavenger. (C) Both the higher molecular mass form of ROMK1 and the lower molecular mass form were detected in lysate from whole kidney, whereas only the higher molecular mass form of ROMK1 was detected in apical membrane protein extract from in situ biotinylated renal tubules. (D) Treatment of apical membrane protein extract of renal tubules with 100 mM DTT decreased the higher molecular mass form and increased the lower molecular mass form of ROMK1. ( E ) Summary plots of the two forms of ROMK1 in apical membranes of renal tubules in the absence or presence of DTT. Methods for preparing cell lysates are the same for mpkCCD c14 cells and kidney tissue. Each experiment was repeated three times and showed consistent results. DTT, dithiothreitol.
    Figure Legend Snippet: ROMK1 on Western blots runs at a higher molecular mass than expected in cultured mpkCCDc14 cells. (A) Western blots from mpkCCD c14 cells or kidney tissue of wild-type (WT) or ROMK knockout (KO) mice. (Left panel) ROMK1 channels in the same gel were first probed with a commercial ROMK1 antibody from Alomone Laboratories (Alomone Ab), and then, the membrane was completely stripped and reprobed with another ROMK1 antibody provided by Paul Welling at the University of Maryland Medical School (R79 AP-4 Ab). (Right panel) ROMK1 channels in a different gel were also detected with R79 AP-4 Ab. (B) Western blot from either control mpkCCD c14 cells or the cells treated for 24 hours with 250 μ M TEMPOL, a superoxide dismutase mimetic and ROS scavenger. (C) Both the higher molecular mass form of ROMK1 and the lower molecular mass form were detected in lysate from whole kidney, whereas only the higher molecular mass form of ROMK1 was detected in apical membrane protein extract from in situ biotinylated renal tubules. (D) Treatment of apical membrane protein extract of renal tubules with 100 mM DTT decreased the higher molecular mass form and increased the lower molecular mass form of ROMK1. ( E ) Summary plots of the two forms of ROMK1 in apical membranes of renal tubules in the absence or presence of DTT. Methods for preparing cell lysates are the same for mpkCCD c14 cells and kidney tissue. Each experiment was repeated three times and showed consistent results. DTT, dithiothreitol.

    Techniques Used: Western Blot, Cell Culture, Knock-Out, Mouse Assay, In Situ

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