Structured Review

Upstate Biotechnology Inc k atpase
Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the <t>α</t> subunit of the Na + ,K + <t>-ATPase</t> was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.
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Images

1) Product Images from "Rat pancreas secretes particulate ecto-nucleotidase CD39"

Article Title: Rat pancreas secretes particulate ecto-nucleotidase CD39

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2003.049411

Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the α subunit of the Na + ,K + -ATPase was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.
Figure Legend Snippet: Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the α subunit of the Na + ,K + -ATPase was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.

Techniques Used: CCK-8 Assay, Western Blot, Positive Control

2) Product Images from "Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis"

Article Title: Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.042689399

Colocalization of subunits of ENaC and Na + -, K + -ATPase with markers specific for TI and TII cells. The preparations of isolated cells used in these experiments contain not only TI cells and TII cells, but various other lung cells liberated by enzymatic digestion, including airway cells, macrophages, and cells from the vascular and interstitial compartments. Immunocytochemistry was performed as described in Methods . The same field of cells is shown in each row, stained with three antibodies and three different fluorophores specific for each antibody. The colors shown in each column are the result of scanning with the appropriate laser for each fluorophore. In the various columns, one can appreciate staining for ( a ) antibodies against various channel or pump proteins (blue color); ( b ) RTI40, a marker for TI cells (red color); and ( c ) RTII70, a marker for TII cells (green color). Column d shows a composite image, created by superimposition of the three separate images shown in columns a , b , and c . Arrows indicate one representative TI cell, one TII cell, and a cell that is neither a TI nor a TII cell. Every TI and TII cell expressed all of the pump/channel proteins. This fact can be appreciated most easily when one looks in column d at the spectral shifts caused by overlapping red (TI) or green (TII) markers with the blue (pump/channel) protein. In this composite image, colocalization of a transport protein subunit (ENaC or Na + -, K + -ATPase) with either TI or TII cell plasma membrane causes a color shift, indicating colocalization of the protein subunit with TI or TII plasma membranes. For example, combination of blue (subunit) with red (TI) is pinkish purple, whereas a combination of blue (subunit) with green (TII) is bluish green. Specific antibodies are labeled in rows 1–5: row 1, αENaC; row 2, βENaC; row 3, γENaC; row 4, α 1 -Na + -, K + -ATPase; row 5, β 1 -Na + -, K + -ATPase; row 6, Cos7 cells stained for αENaC; and row 7, no primary antibody control panel with mixed lung cells.
Figure Legend Snippet: Colocalization of subunits of ENaC and Na + -, K + -ATPase with markers specific for TI and TII cells. The preparations of isolated cells used in these experiments contain not only TI cells and TII cells, but various other lung cells liberated by enzymatic digestion, including airway cells, macrophages, and cells from the vascular and interstitial compartments. Immunocytochemistry was performed as described in Methods . The same field of cells is shown in each row, stained with three antibodies and three different fluorophores specific for each antibody. The colors shown in each column are the result of scanning with the appropriate laser for each fluorophore. In the various columns, one can appreciate staining for ( a ) antibodies against various channel or pump proteins (blue color); ( b ) RTI40, a marker for TI cells (red color); and ( c ) RTII70, a marker for TII cells (green color). Column d shows a composite image, created by superimposition of the three separate images shown in columns a , b , and c . Arrows indicate one representative TI cell, one TII cell, and a cell that is neither a TI nor a TII cell. Every TI and TII cell expressed all of the pump/channel proteins. This fact can be appreciated most easily when one looks in column d at the spectral shifts caused by overlapping red (TI) or green (TII) markers with the blue (pump/channel) protein. In this composite image, colocalization of a transport protein subunit (ENaC or Na + -, K + -ATPase) with either TI or TII cell plasma membrane causes a color shift, indicating colocalization of the protein subunit with TI or TII plasma membranes. For example, combination of blue (subunit) with red (TI) is pinkish purple, whereas a combination of blue (subunit) with green (TII) is bluish green. Specific antibodies are labeled in rows 1–5: row 1, αENaC; row 2, βENaC; row 3, γENaC; row 4, α 1 -Na + -, K + -ATPase; row 5, β 1 -Na + -, K + -ATPase; row 6, Cos7 cells stained for αENaC; and row 7, no primary antibody control panel with mixed lung cells.

Techniques Used: Isolation, Immunocytochemistry, Staining, Marker, Labeling

3) Product Images from "Heterotrimeric G protein subunits are located on rat liver endosomes"

Article Title: Heterotrimeric G protein subunits are located on rat liver endosomes

Journal: BMC Physiology

doi: 10.1186/1472-6793-4-1

Na, K-ATPase as membrane marker. A) Quantitative distribution of α1 and β1 subunits of Na, K-ATPase in different cell fractions: CM (white bars), BLM (gray bars), liver homogenate (dotted bars), RRC (black bars), CURL (down slashed bars), MVB (up slashed bars) and lysosomes (stripped bars). Optical densities were normalized to values in CM as in Figure 1 . Bars represent the mean ± SEM of results from
Figure Legend Snippet: Na, K-ATPase as membrane marker. A) Quantitative distribution of α1 and β1 subunits of Na, K-ATPase in different cell fractions: CM (white bars), BLM (gray bars), liver homogenate (dotted bars), RRC (black bars), CURL (down slashed bars), MVB (up slashed bars) and lysosomes (stripped bars). Optical densities were normalized to values in CM as in Figure 1 . Bars represent the mean ± SEM of results from "n" different preparations of each cell fraction, as indicated on the figure. Both subunits were disenriched in endocytic vesicles and lysosomes compared to CM (p

Techniques Used: Marker

4) Product Images from "Altered Expression of Sodium Transporters and Water Channels in the Submandibular Gland of Rats Treated with Nitric Oxide Synthesis Inhibitors"

Article Title: Altered Expression of Sodium Transporters and Water Channels in the Submandibular Gland of Rats Treated with Nitric Oxide Synthesis Inhibitors

Journal: Electrolytes & Blood Pressure : E & BP

doi: 10.5049/EBP.2008.6.1.9

Expression of α1- and β1-subunits of Na + ,K + -ATPase in the submandibular gland. Legends as in Fig. 1 . Mean±SEM of 6 rats each. ** p
Figure Legend Snippet: Expression of α1- and β1-subunits of Na + ,K + -ATPase in the submandibular gland. Legends as in Fig. 1 . Mean±SEM of 6 rats each. ** p

Techniques Used: Expressing

Related Articles

Transduction:

Article Title: Altered Expression of Sodium Transporters and Water Channels in the Submandibular Gland of Rats Treated with Nitric Oxide Synthesis Inhibitors
Article Snippet: .. The antibodies used were polyclonal anti-rabbit α-1 and β-1 subunits of Na+ ,K+ -ATPase (1:2,500, Upstate Biotechnology, Lake Placid, NY, USA), NKCC2 (1:1,000, Chemicon, Temecula, CA, USA), NHE1 (1:500, Alpha Diagnostic, San Antonio, TX, USA), α-subunit of ENaC (1:500, Alpha Diagnostic, San Antonio, TX, USA), AQP1 (1:1,000, Alomone, Jerusalem, Israel), AQP5 (1:1000, Alpha Diagnostic, San Antonio, TX, USA), endothelial NOS (eNOS) and neuronal NOS (nNOS) (1:750, Transduction; Lexington, KY, USA). β-Actin was used as internal control (1:1,000, Sigma, St. Louis, MO, USA). .. The membrane was then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:1,200) in 2% nonfat milk in TBST for 2 h. The bound secondary antibody was detected by enhanced chemiluminescence (Amersham, Buckinghamshire, England).

Western Blot:

Article Title: Rat pancreas secretes particulate ecto-nucleotidase CD39
Article Snippet: .. In order to check that the 78 kDa band was not due to the plasma membrane from damaged cells that were shed into the juice during collections, we performed a Western blot with the antibody against the α subunit of the Na+ ,K+ -ATPase. .. The pancreatic tissue was positive and showed the predicted band for the intact Na+ ,K+ -ATPase at 110 kDa ( ).

other:

Article Title: β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia
Article Snippet: Monoclonal antibodies were purchased from the following vendors: β1 subunit of Na+ ‐K+ ATPase from Upstate Biotechnology; anti‐glutathione from Virogen, eNOS antibody from Sigma‐Aldrich, and p47phox , phosphorylated eNOS116, phosphorylated eNOS1177, neuronal NOS (nNOS), and α tubulin from Santa Cruz Biotechnology.

Article Title: Impact of Manganese on and Transfer across Blood-Brain and Blood-Cerebrospinal Fluid Barrier in Vitro *
Article Snippet: After an additional 48 h postincubation with CaCl2 , Na+ ,K+ -ATPase is located again at the luminal surface ( BIV ).

Activity Assay:

Article Title: Rat pancreas secretes particulate ecto-nucleotidase CD39
Article Snippet: .. Since ouabain and levamizole were included in the samples, the activity was not due to the Na+ ,K+ -ATPase or alkaline phosphatase. ..

Expressing:

Article Title: Altered Expression of Sodium Transporters and Water Channels in the Submandibular Gland of Rats Treated with Nitric Oxide Synthesis Inhibitors
Article Snippet: .. On the contrary, the expression of α1-subunit of Na+ ,K+ -ATPase was significantly increased, although that of β1-subunit remained unchanged ( ). .. The expression of NKCC2, NHE1, and α-subunit of ENaC was increased significantly ( ).

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    Upstate Biotechnology Inc na k atpase
    Ser163 modulates interaction with <t>Na,K-ATPase.</t> Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,
    Na K Atpase, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc na k atpase α 1 subunit mouse monoclonal
    M 4 returns to the cell surface after agonist stimulation and washout. To selectively examine the amount of M 4 that recycles to the cell surface after CCh treatment and washout, cells were treated with cycloheximide to prevent new receptor synthesis, and cycloheximide was included in all subsequent drug treatments. A , Cells were untreated, treated continuously with CCh for 60 min, or treated with CCh for 60 min, rinsed, and incubated in media alone for 60 min to allow M 4 recovery to the cell surface. In untreated cells, M 4 ( red ) localized primarily to the cell surface, but a small proportion of receptors also showed an intracellular localization. Cell surface M 4 colocalized ( yellow in the merged image) with the plasma membrane marker Na + /K + <t>ATPase</t> ( green ). The inset shows a higher-magnification image of the cell surface, demonstrating that, although M 4 showed colocalization with the Na + /K + ATPase ( yellow ), subdomains exist at the cell surface that contain M 4 but not the Na + /K + ATPase ( red ). Although all cells show staining for the Na + /K + ATPase, not all PC12 cells show M 4 staining, demonstrating heterogeneity of M 4 receptor expression in PC12 cells. After 60 min continual CCh treatment, M 4 trafficked from the cell surface to perinuclear endosomes and no longer colocalized with Na + /K + ATPase. After 60 min CCh treatment followed by 60 min washout, M 4 returned to the cell surface, in which it colocalized with the Na + /K + ATPase. A proportion of receptors also showed an intracellular distribution. Scale bars, 10 μm. B , Measurements of M 4 recycling using quantitative immunocytochemistry were compared with measurements of mAChR recycling using 3 H-NMS. Cells were treated with CCh, rinsed, and incubated in media alone for 60 or 90 min. The amounts of cell surface receptors measured using both assays after CCh washout were normalized to the amounts of cell surface receptors in untreated cells ( black bars and cross-hatched bars ). When expressed as a percentage of untreated cells, the extent of M 4 recycling measured by quantitative immunocytochemistry was slightly higher than the extent of recycling measured by binding assays. Subtracting the residual M 4 remaining after 60 min CCh treatment, however, produced values for M 4 recycling using confocal images ( gray bars ) that were similar to measurements of mAChR recycling using binding assays. This method of calculating M 4 recycling in confocal images was thus used throughout the remainder of the paper. C , The amount of M 4 recovery to the cell was quantified using confocal images at various time points after CCh washout. The graph demonstrates that M 4 began to return to the cell surface as early as 15 min after agonist washout, and, 3 hr after washout, the majority of M 4 (visible by immunocytochemistry) localized to the cell surface.
    Na K Atpase α 1 Subunit Mouse Monoclonal, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ser163 modulates interaction with Na,K-ATPase. Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,

    Journal:

    Article Title: S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells

    doi: 10.1111/j.1524-475X.2008.00432.x

    Figure Lengend Snippet: Ser163 modulates interaction with Na,K-ATPase. Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,

    Article Snippet: Although a primary function of the Na,K-ATPase is to maintain the monovalent cation balance between the interior and the exterior of the cell, it is also a critical participant in establishing polarity of epithelial cells and in cell motility.

    Techniques: FLAG-tag

    FXYD5 expression alters vimentin but not RhoA or Na,K-ATPase expression in LA4 airway cell membrane preparations. (A) Representative GFP fluorescent images of murine LA4 airway cells transfected with pKCEREGFP, demonstrating > 80% transfection

    Journal:

    Article Title: S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells

    doi: 10.1111/j.1524-475X.2008.00432.x

    Figure Lengend Snippet: FXYD5 expression alters vimentin but not RhoA or Na,K-ATPase expression in LA4 airway cell membrane preparations. (A) Representative GFP fluorescent images of murine LA4 airway cells transfected with pKCEREGFP, demonstrating > 80% transfection

    Article Snippet: Although a primary function of the Na,K-ATPase is to maintain the monovalent cation balance between the interior and the exterior of the cell, it is also a critical participant in establishing polarity of epithelial cells and in cell motility.

    Techniques: Expressing, Transfection

    A : representative immunoblots for α-1 Na-K-ATPase in cortex (CTXH) and outer medullary (OMH) homogenates from young male (M) and female (F), control (C) and ANG II (A)-treated mice, as labeled. B : densitometry summary ( n = 6 mice/group). C : representative

    Journal:

    Article Title: Sex differences in adaptive downregulation of pre-macula densa sodium transporters with ANG II infusion in mice

    doi: 10.1152/ajprenal.00088.2009

    Figure Lengend Snippet: A : representative immunoblots for α-1 Na-K-ATPase in cortex (CTXH) and outer medullary (OMH) homogenates from young male (M) and female (F), control (C) and ANG II (A)-treated mice, as labeled. B : densitometry summary ( n = 6 mice/group). C : representative

    Article Snippet: We used a commercial monoclonal antibody against the α-1 subunit of Na-K-ATPase (Upstate Biotechnology, Lake Placid, NY).

    Techniques: Western Blot, Mouse Assay, Labeling

    Altered distribution of the Na,K-ATPase at the sarcolemma of mdx muscle. Unfixed quadriceps muscles of wild-type (A–C, J–M) and mdx (D–I, N–Q) mice were snap frozen and sectioned on a cryostat. Experimental samples (A–I) were double-labeled with affinity-purified chicken antibodies to β-spectrin (B, E, and H) and polyclonal rabbit antibodies to the α1 (A and D) or α2 (G) subunits of the Na,K-ATPase. In color composite images, the α1 or α2 subunits are represented in red, β-spectrin in green, and sites containing both proteins in yellow. (A–C) β-Spectrin and the α1 subunit of the Na,K-ATPase colocalize in costameres in control muscle fibers. Similar results were obtained with the α2 subunit of the Na,K-ATPase (not shown). (D–I) The distribution of the α1 (D) and α2 (G) subunits of the Na,K-ATPase at the sarcolemma is altered, but these subunits still colocalize with the β-spectrin (yellow structures in F and I). (J–Q) Controls were double-labeled either with nonimmune chicken IgY (K and O) and rabbit antibodies to the α1 subunit of the Na,K-ATPase (J and N), or with normal rabbit serum (L and P) and chicken anti–β-spectrin (M and Q). No nonspecific labeling or bleedthrough of fluorescence was observed. Bars, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice

    doi:

    Figure Lengend Snippet: Altered distribution of the Na,K-ATPase at the sarcolemma of mdx muscle. Unfixed quadriceps muscles of wild-type (A–C, J–M) and mdx (D–I, N–Q) mice were snap frozen and sectioned on a cryostat. Experimental samples (A–I) were double-labeled with affinity-purified chicken antibodies to β-spectrin (B, E, and H) and polyclonal rabbit antibodies to the α1 (A and D) or α2 (G) subunits of the Na,K-ATPase. In color composite images, the α1 or α2 subunits are represented in red, β-spectrin in green, and sites containing both proteins in yellow. (A–C) β-Spectrin and the α1 subunit of the Na,K-ATPase colocalize in costameres in control muscle fibers. Similar results were obtained with the α2 subunit of the Na,K-ATPase (not shown). (D–I) The distribution of the α1 (D) and α2 (G) subunits of the Na,K-ATPase at the sarcolemma is altered, but these subunits still colocalize with the β-spectrin (yellow structures in F and I). (J–Q) Controls were double-labeled either with nonimmune chicken IgY (K and O) and rabbit antibodies to the α1 subunit of the Na,K-ATPase (J and N), or with normal rabbit serum (L and P) and chicken anti–β-spectrin (M and Q). No nonspecific labeling or bleedthrough of fluorescence was observed. Bars, 5 μm.

    Article Snippet: Polyclonal rabbit antibodies to a fusion protein containing residues 335–519 of the α1 subunit of the Na,K-ATPase and to a fusion protein containing residues 338–519 of the α2 subunit of the Na,K-ATPase (Upstate Biotechnologies) were used at 3 μg/ml.

    Techniques: Mouse Assay, Labeling, Affinity Purification, Fluorescence

    M 4 returns to the cell surface after agonist stimulation and washout. To selectively examine the amount of M 4 that recycles to the cell surface after CCh treatment and washout, cells were treated with cycloheximide to prevent new receptor synthesis, and cycloheximide was included in all subsequent drug treatments. A , Cells were untreated, treated continuously with CCh for 60 min, or treated with CCh for 60 min, rinsed, and incubated in media alone for 60 min to allow M 4 recovery to the cell surface. In untreated cells, M 4 ( red ) localized primarily to the cell surface, but a small proportion of receptors also showed an intracellular localization. Cell surface M 4 colocalized ( yellow in the merged image) with the plasma membrane marker Na + /K + ATPase ( green ). The inset shows a higher-magnification image of the cell surface, demonstrating that, although M 4 showed colocalization with the Na + /K + ATPase ( yellow ), subdomains exist at the cell surface that contain M 4 but not the Na + /K + ATPase ( red ). Although all cells show staining for the Na + /K + ATPase, not all PC12 cells show M 4 staining, demonstrating heterogeneity of M 4 receptor expression in PC12 cells. After 60 min continual CCh treatment, M 4 trafficked from the cell surface to perinuclear endosomes and no longer colocalized with Na + /K + ATPase. After 60 min CCh treatment followed by 60 min washout, M 4 returned to the cell surface, in which it colocalized with the Na + /K + ATPase. A proportion of receptors also showed an intracellular distribution. Scale bars, 10 μm. B , Measurements of M 4 recycling using quantitative immunocytochemistry were compared with measurements of mAChR recycling using 3 H-NMS. Cells were treated with CCh, rinsed, and incubated in media alone for 60 or 90 min. The amounts of cell surface receptors measured using both assays after CCh washout were normalized to the amounts of cell surface receptors in untreated cells ( black bars and cross-hatched bars ). When expressed as a percentage of untreated cells, the extent of M 4 recycling measured by quantitative immunocytochemistry was slightly higher than the extent of recycling measured by binding assays. Subtracting the residual M 4 remaining after 60 min CCh treatment, however, produced values for M 4 recycling using confocal images ( gray bars ) that were similar to measurements of mAChR recycling using binding assays. This method of calculating M 4 recycling in confocal images was thus used throughout the remainder of the paper. C , The amount of M 4 recovery to the cell was quantified using confocal images at various time points after CCh washout. The graph demonstrates that M 4 began to return to the cell surface as early as 15 min after agonist washout, and, 3 hr after washout, the majority of M 4 (visible by immunocytochemistry) localized to the cell surface.

    Journal: The Journal of Neuroscience

    Article Title: Rab11a and Myosin Vb Regulate Recycling of the M4Muscarinic Acetylcholine Receptor

    doi: 10.1523/JNEUROSCI.22-22-09776.2002

    Figure Lengend Snippet: M 4 returns to the cell surface after agonist stimulation and washout. To selectively examine the amount of M 4 that recycles to the cell surface after CCh treatment and washout, cells were treated with cycloheximide to prevent new receptor synthesis, and cycloheximide was included in all subsequent drug treatments. A , Cells were untreated, treated continuously with CCh for 60 min, or treated with CCh for 60 min, rinsed, and incubated in media alone for 60 min to allow M 4 recovery to the cell surface. In untreated cells, M 4 ( red ) localized primarily to the cell surface, but a small proportion of receptors also showed an intracellular localization. Cell surface M 4 colocalized ( yellow in the merged image) with the plasma membrane marker Na + /K + ATPase ( green ). The inset shows a higher-magnification image of the cell surface, demonstrating that, although M 4 showed colocalization with the Na + /K + ATPase ( yellow ), subdomains exist at the cell surface that contain M 4 but not the Na + /K + ATPase ( red ). Although all cells show staining for the Na + /K + ATPase, not all PC12 cells show M 4 staining, demonstrating heterogeneity of M 4 receptor expression in PC12 cells. After 60 min continual CCh treatment, M 4 trafficked from the cell surface to perinuclear endosomes and no longer colocalized with Na + /K + ATPase. After 60 min CCh treatment followed by 60 min washout, M 4 returned to the cell surface, in which it colocalized with the Na + /K + ATPase. A proportion of receptors also showed an intracellular distribution. Scale bars, 10 μm. B , Measurements of M 4 recycling using quantitative immunocytochemistry were compared with measurements of mAChR recycling using 3 H-NMS. Cells were treated with CCh, rinsed, and incubated in media alone for 60 or 90 min. The amounts of cell surface receptors measured using both assays after CCh washout were normalized to the amounts of cell surface receptors in untreated cells ( black bars and cross-hatched bars ). When expressed as a percentage of untreated cells, the extent of M 4 recycling measured by quantitative immunocytochemistry was slightly higher than the extent of recycling measured by binding assays. Subtracting the residual M 4 remaining after 60 min CCh treatment, however, produced values for M 4 recycling using confocal images ( gray bars ) that were similar to measurements of mAChR recycling using binding assays. This method of calculating M 4 recycling in confocal images was thus used throughout the remainder of the paper. C , The amount of M 4 recovery to the cell was quantified using confocal images at various time points after CCh washout. The graph demonstrates that M 4 began to return to the cell surface as early as 15 min after agonist washout, and, 3 hr after washout, the majority of M 4 (visible by immunocytochemistry) localized to the cell surface.

    Article Snippet: The following primary antibodies were used: early endosome autoantigen 1 (EEA1) mouse monoclonal (1:250; Transduction Laboratories, Lexington KY); M4 mouse monoclonal (1:500; Chemicon, Temecula, CA) ( ) or rabbit polyclonal (0.5 μg/ml) ( ); Na+ /K+ ATPase α-1 subunit mouse monoclonal (1:500; Upstate Biotechnology, Lake Placid, NY); Rab4 rabbit polyclonal (1:500; Biosource, Camarillo, CA), Rab7 rabbit polyclonal (1:100) , or Rab11 rabbit polyclonal (1:500; Zymed, San Francisco, CA); and TfnR mouse monoclonal (1:500; Zymed).

    Techniques: Incubation, Marker, Staining, Expressing, Immunocytochemistry, Binding Assay, Produced