Journal: FEBS letters

Article Title: Intrinsic Mitotic Activity Supports the Human Salivary Gland Acinar Cell Population

doi: 10.1002/1873-3468.13611

Figure Lengend Snippet: Adult human salivary gland tissue sections were co-stained for markers of cell cycle activity and cell type-specific markers to identify mitotically active populations. (A) Human parotid gland (hPG) stained for the nuclear acinar cell marker, MIST1 (red), and the proliferation marker, Ki67 (green). (B) Human submandibular gland (hSMG) stained for MIST1 (red), and Ki67 (green). (C) hPG stained for the acinar cell membrane protein, NKCC1 (red), and Ki67 (green). (D) hSMG stained for NKCC1 (red), and Ki67 (green). (E) hPG stained for the acinar cell membrane marker, AQP3 (red), and Ki67 (green). (F) hSMG stained for the acinar cell membrane marker, AQP3 (red), and Ki67 (green). (G) hPG stained for the acinar cell membrane marker NKCC1 (green) and the proliferation marker, phosphorylated histone H3 (PHH3) (red). (H) hSMG stained for the acinar cell membrane marker NKCC1 (green) and the proliferation marker, PHH3 (red). White asterisks indicate Ki67+ or PHH3+ acinar cells. Nuclei are stained with DAPI (blue). Scale bars =50 μm.

Article Snippet: Primary antibodies, NKCC1 (1:100 goat SC-21545, 1:250 rabbit CST 85403), Mist1 (1:250 rabbit Abcam ab187978), E-Cadherin (1:250 BD 610181), phospho-histone H3 (1:500 or 1:1000 rabbit Millipore 06–570), aurora-B kinase (1:500 or 1:1000 rabbit Abcam ab2254), and Ki67 (1:500 mouse BD 550609) were applied overnight at 4°C.

Techniques: Staining, Activity Assay, Marker, Membrane

Journal: FEBS letters

Article Title: Intrinsic Mitotic Activity Supports the Human Salivary Gland Acinar Cell Population

doi: 10.1002/1873-3468.13611

Figure Lengend Snippet: Human salivary gland tissue was stained for aurora kinase B (AURKB) (red), which localizes between two recently divided cells at the cleavage site shortly after mitosis, and epithelial cadherin (E-CAD) (green) or acinar cell membrane markers, NKCC1 or AQP3 (green). (A) hPG stained for AURKB (red) and E-CAD (green). (B) hSMG stained for AURKB (red) and E-CAD (green). (C) hPG stained for AURKB (red) and the acinar cell marker, NKCC1 (green). (D) hPG stained for AURKB (red) and the acinar cell marker, AQP3 (green). Inserts show 4X magnified AURKB foci between cells. Scale bars = 10 μm.

Article Snippet: Primary antibodies, NKCC1 (1:100 goat SC-21545, 1:250 rabbit CST 85403), Mist1 (1:250 rabbit Abcam ab187978), E-Cadherin (1:250 BD 610181), phospho-histone H3 (1:500 or 1:1000 rabbit Millipore 06–570), aurora-B kinase (1:500 or 1:1000 rabbit Abcam ab2254), and Ki67 (1:500 mouse BD 550609) were applied overnight at 4°C.

Techniques: Staining, Membrane, Marker

Journal: FEBS letters

Article Title: Intrinsic Mitotic Activity Supports the Human Salivary Gland Acinar Cell Population

doi: 10.1002/1873-3468.13611

Figure Lengend Snippet: (A) Quantification of Ki67+ NKCC1+ acinar cells in hPG displayed as a percentage of the total NKCC1+ cell population. (B) Quantification of Ki67+ NKCC1+ acinar cells in hSMG displayed as a percentage of the total NKCC1+ cell population. Dots indicate the percentage of Ki67+ NKCC1+ cells from one of 5 images from three individual slides per sample. Circles represent sample averages from 15 total images with 95% confidence intervals.

Article Snippet: Primary antibodies, NKCC1 (1:100 goat SC-21545, 1:250 rabbit CST 85403), Mist1 (1:250 rabbit Abcam ab187978), E-Cadherin (1:250 BD 610181), phospho-histone H3 (1:500 or 1:1000 rabbit Millipore 06–570), aurora-B kinase (1:500 or 1:1000 rabbit Abcam ab2254), and Ki67 (1:500 mouse BD 550609) were applied overnight at 4°C.

Techniques:

Journal: FEBS letters

Article Title: Intrinsic Mitotic Activity Supports the Human Salivary Gland Acinar Cell Population

doi: 10.1002/1873-3468.13611

Figure Lengend Snippet: (A) Human PG incubated in explant culture with EdU for 24 hours, fixed and stained for EdU (green) and the acinar cell marker, NKCC1 (red). (B) Human SMG incubated with EdU in explant culture and stained for EdU (green) and the acinar cell marker, NKCC1 (red). White asterisks indicate EdU+ acinar cells. (C) Quantification of EdU+ NKCC1+ acinar cells in hPG and hSMG explant cultures displayed as a percentage of the total NKCC1+ cell population. Dots indicate the percentage of EdU+ NKCC1+ cells from one of 5 images from three individual slides per sample. Circles represent sample averages from 15 total images with 95% confidence intervals. Scale bars = 50 μm.

Article Snippet: Primary antibodies, NKCC1 (1:100 goat SC-21545, 1:250 rabbit CST 85403), Mist1 (1:250 rabbit Abcam ab187978), E-Cadherin (1:250 BD 610181), phospho-histone H3 (1:500 or 1:1000 rabbit Millipore 06–570), aurora-B kinase (1:500 or 1:1000 rabbit Abcam ab2254), and Ki67 (1:500 mouse BD 550609) were applied overnight at 4°C.

Techniques: Incubation, Staining, Marker

Journal: Journal of Biological Chemistry

Article Title: Role for Protein Phosphatase 2A in the Regulation of Calu-3 EpithelialNa+-K+-2Cl–, Type 1 Co-transportFunction

doi: 10.1074/jbc.m504473200

Figure Lengend Snippet: FIG. 1. Okadaic acid stimulates NKCC1 activity. A, Calu-3 cells were grown to confluence on filter inserts, se- rum-deprived overnight, and incubated without (vehicle) or with 1 nM okadaic acid for 30 min at 32 °C. Uptake of 86Rb was measured for a 4-min time period. In cells not treated with okadaic acid, base- line bumetanide-sensitive transport of 21.0 3.8 (n 6) nmol/mg protein in- creased to 75.5 14.2 (n 5) nmol/mg protein after stimulation with 10 M me- thoxamine (**, p 0.001). Pretreatment with okadaic acid alone increased base- line NKCC1 activity to 37.4 4.3 (n 3) nmol/mg protein (*, p 0.02) and aug- mented methoxamine-stimulated NKCC1 activity to 113.8 16.7 (n 5) nmol/mg protein. B, dose-response effect of okadaic acid in Calu-3 cells. Cells were pretreated with varying concentrations of okadaic acid for 30 min at 32 °C, and uptake of 86Rb was measured as in A. Increasing concentrations of okadaic acid stimulated bumetanide-sensitive 86Rb uptake to maximal levels at 3–10 nM. Levels of sig- nificance: *, p 0.05; **, p 0.002; and ***, p 0.0001. C, calculation of EC50 for the stimulation of NKCC1 activity by oka- daic acid. Non-linear regression analysis of normalized transport data versus log okadaic acid concentration yielded a log EC50 of 0.37 nM or an EC50 of 0.42 nM with a Hill slope of 0.64.

Article Snippet: Mouse polyclonal antibodies to full-length human PP1 (E-9) and to human PP2B catalytic subunit, goat polyclonal antibody to NKCC1 (N-16), polyclonal anti-GST antibody, anti-GST-agarose beads, and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Incubation, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Role for Protein Phosphatase 2A in the Regulation of Calu-3 EpithelialNa+-K+-2Cl–, Type 1 Co-transportFunction

doi: 10.1074/jbc.m504473200

Figure Lengend Snippet: FIG. 3. Expression of serine-threonine protein phosphatases in Calu-3 cells. Calu-3 cells were grown to confluence and serum- deprived overnight. TCL were used directly for immunoblot analysis (IB) in 20-g aliquots or for immunoprecipitation of NKCC1 (B), PP1 (C), or PP2A (D) followed by immunoblot analysis for the indicated proteins. For immunoblot analysis, proteins were separated on 4–15% gradient slab gels and transferred to PVDF membrane paper. Mem- brane strips were washed extensively in blocking buffer and immuno- blotted with antibodies directed against a protein of interest. A, immu- noblot analysis for protein phosphatase subtypes. PP1 was detected using mouse anti-PP1 at 1:1000 dilution, and PP2A was detected using anti-PP2 catalytic subunit at a 1:2500 dilution or a PP2A clone 1D6 at a 1:1000 dilution and PP2C/ at a 1:1000 dilution. Protein bands immunoreactive to antibody to PP1 and PP2A were detected at 39- and 36-kDa protein bands, respectively, and to antibody to PP2C at 45 kDa. Results represent a typical immunoblot for samples from at least five separate experiments. B, co-immunoprecipitation of protein phospha- tase enzymes with NKCC1. NKCC1 was immunoprecipitated from TCL as described under “Experimental Procedures” and subjected to SDS- PAGE and immunoblot analysis for the indicated proteins. NKCC1 was detected as a doublet at 170 kDa in TCL and IP of NKCC1. PP2A was detected in TCL and in immunoprecipitates of NKCC1. PP1 and PP2C were detected in TCL but not consistently observed in immunoprecipi- tates of NKCC1. The figure represents a typical immunoblot for sam- ples from at least three separate experiments. C, immunoprecipitation of PP1. Immunoblot analysis for NKCC1 using an affinity-purified monoclonal antibody to NKCC1 revealed a protein band in TCL at 170 kDa in TCL that was not detected in immunoprecipitates of PP1. Control immunoblots for PP1 confirm the presence of PP1 in immuno- precipitates. The figure represents typical results of at least three

Article Snippet: Mouse polyclonal antibodies to full-length human PP1 (E-9) and to human PP2B catalytic subunit, goat polyclonal antibody to NKCC1 (N-16), polyclonal anti-GST antibody, anti-GST-agarose beads, and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Immunoprecipitation, Membrane, Blocking Assay, SDS Page, Affinity Purification, Control

Journal: Journal of Biological Chemistry

Article Title: Role for Protein Phosphatase 2A in the Regulation of Calu-3 EpithelialNa+-K+-2Cl–, Type 1 Co-transportFunction

doi: 10.1074/jbc.m504473200

Figure Lengend Snippet: FIG. 4. Examination of PP1 co-immunoprecipitation with NKCC1. Calu-3 cells were grown to confluence, serum-deprived over- night, and then treated with vehicle of HBSS or 10 M methoxamine for 4 min at 35 °C. After 4 min of incubation, cells were rapidly washed with ice-cold PBS and solubilized in lysis buffer. NKCC1 was immuno- precipitated from TCL as described under “Experimental Procedures” and probed by immunoblot analysis for PP1 or as a control, NKCC1. The upper panel shows that PP1 was detected in TCL but not in immunoprecipitates. Control immunoblots in lower panel show that NKCC1 was detected in TCL and in IP from cells treated with vehicle or with methoxamine. Data are representative of two experiments in which each condition was replicated in triplicate.

Article Snippet: Mouse polyclonal antibodies to full-length human PP1 (E-9) and to human PP2B catalytic subunit, goat polyclonal antibody to NKCC1 (N-16), polyclonal anti-GST antibody, anti-GST-agarose beads, and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Incubation, Lysis, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Role for Protein Phosphatase 2A in the Regulation of Calu-3 EpithelialNa+-K+-2Cl–, Type 1 Co-transportFunction

doi: 10.1074/jbc.m504473200

Figure Lengend Snippet: FIG. 7. Pull down of PP2A by the N terminus of NKCC1. Recom- binant tagged NKCC1-CT and NKCC1-NT were expressed as described under “Experimental Procedures.” TCL were prepared from Calu-3 cells and probed with 30 g of recombinant protein. Protein complexes were recovered using anti-GST antibody coupled to agarose beads and sub- jected to immunoblot analysis for PP2A or, as a control, the GST tag. The GST tag was detected in protein complexes but not in TCL, indi- cating recovery of the recombinant proteins by pull down. PP2A was detected in pull downs with the N terminus of NKCC1 but not the C terminus. Data are representative of two experiments in which each condition was replicated at least three times.

Article Snippet: Mouse polyclonal antibodies to full-length human PP1 (E-9) and to human PP2B catalytic subunit, goat polyclonal antibody to NKCC1 (N-16), polyclonal anti-GST antibody, anti-GST-agarose beads, and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Recombinant, Western Blot, Control