Structured Review

Developmental Studies Hybridoma Bank k atpase
The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
K Atpase, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/k atpase/product/Developmental Studies Hybridoma Bank
Average 92 stars, based on 3 article reviews
Price from $9.99 to $1999.99
k atpase - by Bioz Stars, 2020-05
92/100 stars

Images

1) Product Images from "The Antioxidant Peroxiredoxin 6 (Prdx6) Exhibits Different Profiles in the Livers of Seawater- and Fresh Water-Acclimated Milkfish, Chanos chanos, upon Hypothermal Challenge"

Article Title: The Antioxidant Peroxiredoxin 6 (Prdx6) Exhibits Different Profiles in the Livers of Seawater- and Fresh Water-Acclimated Milkfish, Chanos chanos, upon Hypothermal Challenge

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2016.00580

The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + -ATPase (NKA), and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
Figure Legend Snippet: The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + -ATPase (NKA), and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P

Techniques Used: Western Blot

2) Product Images from "Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation"

Article Title: Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029462

Na + , K + -ATPase protein levels in gill of fish injected with SGK1 vivo-morpholino. Freshwater acclimated fish were injected with 14 µg/g SGK1 or control vivo-morpholino. Four hours after injection fish were transferred to seawater for 1 h. The increase in plasma membrane Na + , K + -ATPase in seawater fish compared to freshwater fish was not due to differences in contamination of the membrane preparation by cytosolic proteins, since the amount of Rab4a in each preparation was minimal, and equivalent in all membrane preparations (see Figure 4A ). A : Representative Western blot of Na + , K + -ATPase. B : Summary of Na + , K + -ATPase WCL protein levels. C : Summary of Na + , K + -ATPase PM protein levels. n = 4. Different letters indicate statistically significant treatment means p
Figure Legend Snippet: Na + , K + -ATPase protein levels in gill of fish injected with SGK1 vivo-morpholino. Freshwater acclimated fish were injected with 14 µg/g SGK1 or control vivo-morpholino. Four hours after injection fish were transferred to seawater for 1 h. The increase in plasma membrane Na + , K + -ATPase in seawater fish compared to freshwater fish was not due to differences in contamination of the membrane preparation by cytosolic proteins, since the amount of Rab4a in each preparation was minimal, and equivalent in all membrane preparations (see Figure 4A ). A : Representative Western blot of Na + , K + -ATPase. B : Summary of Na + , K + -ATPase WCL protein levels. C : Summary of Na + , K + -ATPase PM protein levels. n = 4. Different letters indicate statistically significant treatment means p

Techniques Used: Fluorescence In Situ Hybridization, Injection, Western Blot

3) Product Images from "Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney"

Article Title: Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2003.042903

Double-staining of kidney sections for sgk1, the Na + ,K + -ATPase and actin Staining of a transverse section of the outer medulla for sgk1 ( A ), the α subunit of the Na + ,K + -ATPase ( B ), and overlay of these two images ( C ) showing colocalization of the two proteins. Higher magnification of the outer medulla stained for sgk1 ( D ), and the α subunit of the Na + ,K + -ATPase ( E ) are also shown. Deep infolds of the basolateral membrane are delineated by the two antibodies whereas no fluorescent signal is apparent in the apical membrane. F , section of cortex stained with anti-sgk1. Arrows indicate the basolateral localization of the signal. These tubules correspond to CCT because the staining is restricted to principal cells and absent in intercalated cells. Actin labelled with DNase I conjugated with Texas red ( G ) distributes over the whole cytoplasm with enhancement of apical microvilli. Overlay shows sgk1 in the basolateral membrane but absent from the apical membrane ( H ).
Figure Legend Snippet: Double-staining of kidney sections for sgk1, the Na + ,K + -ATPase and actin Staining of a transverse section of the outer medulla for sgk1 ( A ), the α subunit of the Na + ,K + -ATPase ( B ), and overlay of these two images ( C ) showing colocalization of the two proteins. Higher magnification of the outer medulla stained for sgk1 ( D ), and the α subunit of the Na + ,K + -ATPase ( E ) are also shown. Deep infolds of the basolateral membrane are delineated by the two antibodies whereas no fluorescent signal is apparent in the apical membrane. F , section of cortex stained with anti-sgk1. Arrows indicate the basolateral localization of the signal. These tubules correspond to CCT because the staining is restricted to principal cells and absent in intercalated cells. Actin labelled with DNase I conjugated with Texas red ( G ) distributes over the whole cytoplasm with enhancement of apical microvilli. Overlay shows sgk1 in the basolateral membrane but absent from the apical membrane ( H ).

Techniques Used: Double Staining, Staining

Related Articles

IA:

Article Title: Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
Article Snippet: .. Western blot analysis of CFTR (Clone ACL-006, 1∶500 dilution, Alomone Labs, Jerusalem, Israel), SGK1 (anti-SGK #S5188, 1∶2000 dilution, Sigma-Aldrich, St. Louis, MO), Na+ , K+ -ATPase (Na+ , K+ -ATPase a5 supernatant, 1 µg/ml, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), Rab4a (SC-312, 1∶500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Clone C4, 1∶2000 dilution, MP Biomedicals, Solon, OH) in gill lysates was performed as previously described , . ..

Article Title: The Antioxidant Peroxiredoxin 6 (Prdx6) Exhibits Different Profiles in the Livers of Seawater- and Fresh Water-Acclimated Milkfish, Chanos chanos, upon Hypothermal Challenge
Article Snippet: .. Then, the immunoblotting was performed as described above except that Na+ , K+ -ATPase (NKA; α5, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) was used as the loading control. ..

Article Title: ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo
Article Snippet: .. Monoclonal anti-Na+ ,K+ -ATPase developed by Douglas M. Fambrough was obtained from the Developmental Studies Hybridoma Bank, under the auspices of NICHD, maintained by the University of Iowa (Iowa City, IA, USA). .. Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Pierce (Rockford, IL, USA).

Article Title: Eccentric contractions increase the phosphorylation of tuberous sclerosis complex-2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome
Article Snippet: .. Anti-Na+ ,K+ -ATPase (A6Fs) was purchased from Developmental Studies Hybridoma Bank (Iowa City, IA, USA). .. Peroxidase-conjugated anti-rabbit (PI-1000) and anti-mouse antibodies (PI-2000) were purchased from Vector Laboratories (Burlingame, CA, USA).

Immunoprecipitation:

Article Title: Phosphorylation of Adaptor Protein-2 ?2 Is Essential for Na+,K+-ATPase Endocytosis in Response to Either G Protein-Coupled Receptor or Reactive Oxygen Species
Article Snippet: .. The following antibodies were used: the Na+ ,K+ -ATPase was immunoprecipitated using an antibody against its α-subunit (α5) developed by Dr. Fambrough and obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Department of Biological Sciences. ..

other:

Article Title: Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney
Article Snippet: Monoclonal antibody α5 against the α subunit of the Na+ ,K+ -ATPase, developed by Dr D. Fambrough , was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa, USA.

Article Title: AICAR activates AMPK and alters PIP2 association with the epithelial sodium channel ENaC to inhibit Na+ transport in H441 lung epithelial cells
Article Snippet: However, in five of our biotinylation experiments, a small amount of Na+ ,K+ -ATPase was observed in the biotinylated apical protein fraction.

Western Blot:

Article Title: Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
Article Snippet: .. Western blot analysis of CFTR (Clone ACL-006, 1∶500 dilution, Alomone Labs, Jerusalem, Israel), SGK1 (anti-SGK #S5188, 1∶2000 dilution, Sigma-Aldrich, St. Louis, MO), Na+ , K+ -ATPase (Na+ , K+ -ATPase a5 supernatant, 1 µg/ml, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), Rab4a (SC-312, 1∶500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Clone C4, 1∶2000 dilution, MP Biomedicals, Solon, OH) in gill lysates was performed as previously described , . ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Developmental Studies Hybridoma Bank mouse anti na k atpase nak alpha subunit
    A severe distortion in lumen and cell morphology results when tissue is fixed in PEM (PIPES, EGTA, and MgSO 4 ) buffer. (A–D, K–N) Immunofluorescence micrographs of 96 hours (h) after puparium formation (APF) w 1118 (wild type) Drosophila ommatidium in a cross optical section (A–D) or in a vertical optical section (K–N) showing the positioning of the rhabdomeres and the size of the inter-rhabdomeral space (IRS). Tissues were fixed in PEM buffer (A,B,K,M) or in PBS buffer (C,D,L,N) . The rhabdomeres, F-actin, are labeled with Phalloidin (magenta), and EYS (A,C,K,L) or Na + K + <t>ATPase</t> (NaK) which labels the basolateral membranes (B,D,M,N) are shown in green. (E–F) Quantitative analysis of the area of the IRS (E) or the average area of R1–R7 photoreceptor cells (F) in PEM or PBS buffered conditions as seen in (A–D) . Values represent mean ± SEM. ** P
    Mouse Anti Na K Atpase Nak Alpha Subunit, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti na k atpase nak alpha subunit/product/Developmental Studies Hybridoma Bank
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse anti na k atpase nak alpha subunit - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    93
    Developmental Studies Hybridoma Bank na k atpase
    The immunolabeling pattern of α-subunit of Na + /K + <t>-ATPase</t> α-subunit in the lamina neuropil of repo- Gal4 / + control flies (A) and repo- Gal4 > UAS -DmMANF RNAi flies (B) . In the lamina of control flies (A) , membranes containing the α-subunit of Na + /K + -ATPase are well visible in the localization of epithelial glia and other cells, and therefore cartridges (rectangle-a single cartridge) can easily be discerned. In the lamina of experimental flies with reduced expression of DmMANF in glia (B) , on the other hand, cartridges can no longer be distinguished. Numerous spots of stronger but diffuse fluorescence (arrows) can be observed in the place of punctures. Scale bar: 10 μm. (C) Although the distribution of Na + /K + -ATPase α-subunit is disturbed in the lamina neuropil (Ln), its presence at the basolateral membrane of photoreceptors (arrowhead) of the retina (R) and the expression in the lamina cortex (Lc) are not affected. Degenerative changes are surrounded by membranous aggregates strongly labeled against α-subunit of sodium-potassium pump (arrow). Scale bar: 20 μm.
    Na K Atpase, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/na k atpase/product/Developmental Studies Hybridoma Bank
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    na k atpase - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    88
    Developmental Studies Hybridoma Bank na k atpase polyclonal antibody
    Heterologous protein expression of p.Asp219Val hERG A) Representative Immunoblot shows whole cell lysate of HEK 293 cells transiently transfected with WT hERG, 50/50 mix or p.Asp219Val hERG plasmid DNA. Antibodies targeted against hERG and Na + /K + <t>-ATPase</t> (loading control) were used. hERG channel protein appears as a double band (a discrete 135 kD species and 155 kD broad smear of heavily glycosylated protein) representing immature and mature hERG, respectively. These data are a summary of 5 immunoblots performed and analyzed. B) Histogram showing summary of total hERG protein from 5 immunoblots, normalized to Na + /K + -ATPase. Graph shows densitometry quantification of WT hERG and p.Asp219Val hERG and demonstrates that there was no significant difference between total protein expression. C) Histogram illustrates relative distribution of immature hERG (135 kD) and mature hERG (155 kD), normalized to 1 for WT, 50/50 mix of WT and mutant, and p.Asp219Val hERG. This reflects the ratio of channel protein expressed on the cell membrane to the channel present in the ER/Golgi. The ratio indicates trafficking success to the surface of the cell. There was no significant difference between these ratios for each of the samples.
    Na K Atpase Polyclonal Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/na k atpase polyclonal antibody/product/Developmental Studies Hybridoma Bank
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    na k atpase polyclonal antibody - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    Image Search Results


    A severe distortion in lumen and cell morphology results when tissue is fixed in PEM (PIPES, EGTA, and MgSO 4 ) buffer. (A–D, K–N) Immunofluorescence micrographs of 96 hours (h) after puparium formation (APF) w 1118 (wild type) Drosophila ommatidium in a cross optical section (A–D) or in a vertical optical section (K–N) showing the positioning of the rhabdomeres and the size of the inter-rhabdomeral space (IRS). Tissues were fixed in PEM buffer (A,B,K,M) or in PBS buffer (C,D,L,N) . The rhabdomeres, F-actin, are labeled with Phalloidin (magenta), and EYS (A,C,K,L) or Na + K + ATPase (NaK) which labels the basolateral membranes (B,D,M,N) are shown in green. (E–F) Quantitative analysis of the area of the IRS (E) or the average area of R1–R7 photoreceptor cells (F) in PEM or PBS buffered conditions as seen in (A–D) . Values represent mean ± SEM. ** P

    Journal: BMC Developmental Biology

    Article Title: Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation

    doi: 10.1186/s12861-015-0056-y

    Figure Lengend Snippet: A severe distortion in lumen and cell morphology results when tissue is fixed in PEM (PIPES, EGTA, and MgSO 4 ) buffer. (A–D, K–N) Immunofluorescence micrographs of 96 hours (h) after puparium formation (APF) w 1118 (wild type) Drosophila ommatidium in a cross optical section (A–D) or in a vertical optical section (K–N) showing the positioning of the rhabdomeres and the size of the inter-rhabdomeral space (IRS). Tissues were fixed in PEM buffer (A,B,K,M) or in PBS buffer (C,D,L,N) . The rhabdomeres, F-actin, are labeled with Phalloidin (magenta), and EYS (A,C,K,L) or Na + K + ATPase (NaK) which labels the basolateral membranes (B,D,M,N) are shown in green. (E–F) Quantitative analysis of the area of the IRS (E) or the average area of R1–R7 photoreceptor cells (F) in PEM or PBS buffered conditions as seen in (A–D) . Values represent mean ± SEM. ** P

    Article Snippet: Primary antibodies used in this study were: mouse anti-EYS (mAb 21A6, Developmental Studies Hybridoma Bank, 1:50 for 48 h APF pupae and 1:100 for later staged pupae or adults [ , , ], mouse anti-Na+ K+ ATPase (NaK) alpha subunit (mAb a5, 1:100, Developmental Studies Hybridoma Bank) [ ].

    Techniques: End-sequence Profiling, Immunofluorescence, Labeling

    The immunolabeling pattern of α-subunit of Na + /K + -ATPase α-subunit in the lamina neuropil of repo- Gal4 / + control flies (A) and repo- Gal4 > UAS -DmMANF RNAi flies (B) . In the lamina of control flies (A) , membranes containing the α-subunit of Na + /K + -ATPase are well visible in the localization of epithelial glia and other cells, and therefore cartridges (rectangle-a single cartridge) can easily be discerned. In the lamina of experimental flies with reduced expression of DmMANF in glia (B) , on the other hand, cartridges can no longer be distinguished. Numerous spots of stronger but diffuse fluorescence (arrows) can be observed in the place of punctures. Scale bar: 10 μm. (C) Although the distribution of Na + /K + -ATPase α-subunit is disturbed in the lamina neuropil (Ln), its presence at the basolateral membrane of photoreceptors (arrowhead) of the retina (R) and the expression in the lamina cortex (Lc) are not affected. Degenerative changes are surrounded by membranous aggregates strongly labeled against α-subunit of sodium-potassium pump (arrow). Scale bar: 20 μm.

    Journal: Frontiers in Neuroscience

    Article Title: Downregulation of DmMANF in Glial Cells Results in Neurodegeneration and Affects Sleep and Lifespan in Drosophila melanogaster

    doi: 10.3389/fnins.2017.00610

    Figure Lengend Snippet: The immunolabeling pattern of α-subunit of Na + /K + -ATPase α-subunit in the lamina neuropil of repo- Gal4 / + control flies (A) and repo- Gal4 > UAS -DmMANF RNAi flies (B) . In the lamina of control flies (A) , membranes containing the α-subunit of Na + /K + -ATPase are well visible in the localization of epithelial glia and other cells, and therefore cartridges (rectangle-a single cartridge) can easily be discerned. In the lamina of experimental flies with reduced expression of DmMANF in glia (B) , on the other hand, cartridges can no longer be distinguished. Numerous spots of stronger but diffuse fluorescence (arrows) can be observed in the place of punctures. Scale bar: 10 μm. (C) Although the distribution of Na + /K + -ATPase α-subunit is disturbed in the lamina neuropil (Ln), its presence at the basolateral membrane of photoreceptors (arrowhead) of the retina (R) and the expression in the lamina cortex (Lc) are not affected. Degenerative changes are surrounded by membranous aggregates strongly labeled against α-subunit of sodium-potassium pump (arrow). Scale bar: 20 μm.

    Article Snippet: Interestingly, we found that the distribution of the α-subunit of Na+ /K+ -ATPase in the lamina neuropil was highly disorganized, and the lamina cartridges could no longer be distinguished (Figures ).

    Techniques: Immunolabeling, Expressing, Fluorescence, Labeling

    CDX2 is required for apical–basal polarity in human Caco-2 cells. ( A ) Western blot for CDX2 demonstrates efficient knockdown of CDX2 expression in lentiviral shCDX2 transduced Caco-2 cells (Sh), compared with parental cells (Pr) and nontargeting lentiviral particle-treated control cells (Ct), at first passage after viral transduction. ( B , C ) Control Caco-2 cells form cysts in 3D culture within 72 h, demonstrating a central lumen, while CDX2 knockdown cells fail to form a primary lumen even after 5-d culture. ( D–G ) Cysts were stained for F-actin (red), E-cadherin (green), and DAPI (blue) at various time points. Note that CDX2 knockdown cells elaborate multiple small lumens at 48 h ( E ), but fail to promote a central lumen ( G ). ( H , I ) Cysts were stained for basolateral marker CTNNB1 (β-catenin) (red). ( J , K ) Cysts were stained for a basal transporter, Na + /K + -ATPase (green). ( L , M ) Control and CDX2-deficient Caco-2 cysts were stained for PRKCZ in red. Nuclei were labeled by DAPI in blue. ( N ) In vitro protein kinase C assay demonstrates a reduction of PRKCZ kinase activity in CDX2-deficient cells by 25.6%. Bars, 20 μm.

    Journal: Genes & Development

    Article Title: Cdx2 regulates endo-lysosomal function and epithelial cell polarity

    doi: 10.1101/gad.1921510

    Figure Lengend Snippet: CDX2 is required for apical–basal polarity in human Caco-2 cells. ( A ) Western blot for CDX2 demonstrates efficient knockdown of CDX2 expression in lentiviral shCDX2 transduced Caco-2 cells (Sh), compared with parental cells (Pr) and nontargeting lentiviral particle-treated control cells (Ct), at first passage after viral transduction. ( B , C ) Control Caco-2 cells form cysts in 3D culture within 72 h, demonstrating a central lumen, while CDX2 knockdown cells fail to form a primary lumen even after 5-d culture. ( D–G ) Cysts were stained for F-actin (red), E-cadherin (green), and DAPI (blue) at various time points. Note that CDX2 knockdown cells elaborate multiple small lumens at 48 h ( E ), but fail to promote a central lumen ( G ). ( H , I ) Cysts were stained for basolateral marker CTNNB1 (β-catenin) (red). ( J , K ) Cysts were stained for a basal transporter, Na + /K + -ATPase (green). ( L , M ) Control and CDX2-deficient Caco-2 cysts were stained for PRKCZ in red. Nuclei were labeled by DAPI in blue. ( N ) In vitro protein kinase C assay demonstrates a reduction of PRKCZ kinase activity in CDX2-deficient cells by 25.6%. Bars, 20 μm.

    Article Snippet: Antibodies used for immunohistochemistry and Western blots include Cdx2 (Biogenex), Prkci (BD Transduction Laboratories), Par-3 (07-330, Millipore; 36-2301, Invitrogen), Prkcz (C-20, Santa Cruz Biotechnology), ZO-1 (Invitrogen), Laminin (abcam), Mark2 (Cell Signaling), Cdc42 (Cell Signaling), Par6A (G-9, Santa Cruz Biotechnology), Par6B (M-64, Santa Cruz Biotechnology), Lkb1 (Abcam), E-cadherin and Ctnnb1 (BD Transduction Laboratories), Tubulin (Sigma), Myo5b (Abcam), Myo1a (C-12, Santa Cruz Biotechnology), Myo1e (N-13, Santa Cruz Biotechnology), Myo6 (H-215, Santa Cruz Biotechnology), CaM (FL-149, Santa Cruz Biotechnology), Plastin (H-300, Santa Cruz Biotechnology), Spectrin (C-11, Santa Cruz Biotechnology), Ezrin (Cell Signaling), Rab8A (N-20, Santa Cruz Biotechnology), Rab27a (17817-1-AP, Proteintech Group), Rab11a (R0009, US Biological), EEA1 (Abcam), Lamp1 (Abcam, and 1D4B, Developmental Studies Hybridoma Bank), Lamp2 (Abcam), LC3A/B (Abcam), Catalase (Abcam), Cathepsin D (Abcam), Na+ /K+ -ATPase (a5, Developmental Studies Hybridoma Bank, The University of Iowa), and β-actin (Cell Signaling).

    Techniques: Western Blot, Expressing, Transduction, Staining, Marker, Labeling, In Vitro, Activity Assay

    Heterologous protein expression of p.Asp219Val hERG A) Representative Immunoblot shows whole cell lysate of HEK 293 cells transiently transfected with WT hERG, 50/50 mix or p.Asp219Val hERG plasmid DNA. Antibodies targeted against hERG and Na + /K + -ATPase (loading control) were used. hERG channel protein appears as a double band (a discrete 135 kD species and 155 kD broad smear of heavily glycosylated protein) representing immature and mature hERG, respectively. These data are a summary of 5 immunoblots performed and analyzed. B) Histogram showing summary of total hERG protein from 5 immunoblots, normalized to Na + /K + -ATPase. Graph shows densitometry quantification of WT hERG and p.Asp219Val hERG and demonstrates that there was no significant difference between total protein expression. C) Histogram illustrates relative distribution of immature hERG (135 kD) and mature hERG (155 kD), normalized to 1 for WT, 50/50 mix of WT and mutant, and p.Asp219Val hERG. This reflects the ratio of channel protein expressed on the cell membrane to the channel present in the ER/Golgi. The ratio indicates trafficking success to the surface of the cell. There was no significant difference between these ratios for each of the samples.

    Journal: Human mutation

    Article Title: An interdomain KCNH2 mutation produces an intermediate long QT syndrome

    doi: 10.1002/humu.22805

    Figure Lengend Snippet: Heterologous protein expression of p.Asp219Val hERG A) Representative Immunoblot shows whole cell lysate of HEK 293 cells transiently transfected with WT hERG, 50/50 mix or p.Asp219Val hERG plasmid DNA. Antibodies targeted against hERG and Na + /K + -ATPase (loading control) were used. hERG channel protein appears as a double band (a discrete 135 kD species and 155 kD broad smear of heavily glycosylated protein) representing immature and mature hERG, respectively. These data are a summary of 5 immunoblots performed and analyzed. B) Histogram showing summary of total hERG protein from 5 immunoblots, normalized to Na + /K + -ATPase. Graph shows densitometry quantification of WT hERG and p.Asp219Val hERG and demonstrates that there was no significant difference between total protein expression. C) Histogram illustrates relative distribution of immature hERG (135 kD) and mature hERG (155 kD), normalized to 1 for WT, 50/50 mix of WT and mutant, and p.Asp219Val hERG. This reflects the ratio of channel protein expressed on the cell membrane to the channel present in the ER/Golgi. The ratio indicates trafficking success to the surface of the cell. There was no significant difference between these ratios for each of the samples.

    Article Snippet: The Na+ /K+ -atpase polyclonal antibody used as a control in the immunoblots was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis