Journal: The FASEB Journal

Article Title: Activation of the calcium sensing receptor increases claudin‐14 expression via a PLC ‐p38‐Sp1 pathway

doi: 10.1096/fj.202002137RRR

Figure Lengend Snippet: SP1 inhibits expression of the 1500 bp cldn14 promoter reporter construct. (A) Luciferase activity from the Cldn14 reporter construct after transfection with the transcription factor SP1. SP1 cotransfection significantly decreased luciferase activity of cells cotransfected with Empty Vector (EV) or the CaSR under low and high Ca 2+ concentrations. Ordinary one‐way ANOVA was used to determine statistical difference (*** /### p < .001; ## p < .01; # p < .05). (B) Immunoblots demonstrating co‐expression of the CaSR with SP1. (C) Immunohistochemical staining of a murine renal section for SP1 (brown) and NKCC2 (blue). (D) high magnification image of thick ascending limb with nuclear localization of SP1. (E) Representative immunoblots of myc tagged SP1 run on regular or FeCl 3 ‐loaded gels. (F) Relative phospho‐SP1 protein abundance decreased when cells were incubated in with high [Ca 2+ ] and expressed the CaSR. Friedman test with Dunn's multiple comparisons test were used to determine statistical differences (* p < .05)

Article Snippet: Thereafter, the sections were boiled again in TEG buffer to remove bound antibodies and a second round of incubation with rabbit polyclonal antibodies against NKCC2 was performed (SLC12A1, HPA014967, Sigma‐Aldrich).

Techniques: Expressing, Construct, Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Western Blot, Immunohistochemical staining, Staining, Incubation

Journal: Protein & Cell

Article Title: Vitamin C alleviates aging defects in a stem cell model for Werner syndrome

doi: 10.1007/s13238-016-0278-1

Figure Lengend Snippet: VC represses aging properties in WS MSCs. (A) Growth curve analyzing the population doubling of MSCs. (B) VC promoted proliferation in WRN -/- MSCs. Representative immunofluorescence staining (left) and quantitative analysis (right) of Ki67 in vehicle or VC treated (7 days) WT MSCs and WRN -/- MSCs. Scale bar, 50 µm. (C) H2DCFDA based measurement of reactive oxygen species (ROS) in vehicle or VC treated (7 days) WT MSCs and WRN -/- MSCs. (D) Telomere lengths of WS MSCs with or without VC treatment were measured by quantitative RT-PCR. (E) Expression of senescence-associated proteins p16 Ink4a and GATA4 was examined by Western blot, and quantitative results were shown on the right. (F) ELISA showing a decrease in IL-6 and IL-8 secretion in WRN -/- MSCs after VC treatment. The values were normalized by the cell numbers. Data are represented as mean ± SEM, ** P < 0.01, *** P < 0.001; n ≥ 3

Article Snippet: Abcam: anti-H3K9me3 (ab8898); Santa Cruz Biotechnology: anti-β-actin (SC-130301), anti-GATA4 (SC-1237); Cell Signaling Technology: Phospho-(Ser/Thr) ATM/ATR Substrate Antibody (2851), anti-HP1α (2616), anti-p16 (4828); Millipore: anti-γ-H2AX (05-636); BD Bioscience: anti-LAP2β (611000); Vector: anti-Ki67 (VP-RM04).

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

doi: 10.1038/srep36016

Figure Lengend Snippet: Representative confocal microscope images show extent of expression and subcellular localization of TMEM16A, SLC26A4, carbonic anhydrase 2 (CA2), SLC12A2, and ATP12A (images taken from BE37 cells; similar results were obtained from BE63 cells). Whenever permitted by the combination of primary antibodies, acetylated tubulin and MUC5AC were also stained as markers of ciliated and goblet cells, respectively. Bronchial epithelia were kept under control conditions or treated with IL-4 for 72 hrs. Larger images: xy sections (scale bar: 20 μm). Inset: xz sections (scale bar: 10 μm). Images with a different scale of view are shown in .

Article Snippet: The following primary antibodies and dilutions were used: rabbit monoclonal anti-TMEM16A [SP31] (ab64085, Abcam) at 1:200, mouse IgG1 anti-CFTR (ab570, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics) at 1:250, mouse polyclonal anti-SLC26A4 (H00005172-A01, Abnova) at 1:200, rabbit polyclonal anti-SLC12A2 (HPA020130, Sigma-Aldrich) at 1:1000, rabbit monoclonal anti-CA2 [EPR5195] (ab124687, Abcam) at 1:500, mouse IgG1 anti-MUC5AC (NCL-HGM-45M1, Novocastra) at 1:100, mouse IgG2B anti-acetylated tubulin (7451, Sigma Aldrich) at 1:300, rabbit polyclonal anti-ATP12A (HPA039526, Sigma-Aldrich) at 1:400.

Techniques: Microscopy, Expressing, Staining

Journal: Scientific Reports

Article Title: Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

doi: 10.1038/srep36016

Figure Lengend Snippet: For simplicity, the cartoon shows all channels and transporters within the same cell although some components (e.g. CFTR and TMEM16A) are localized in separate cell types. The NKCC1 transporter (SLC12A2) promotes the intracellular accumulation of Cl − that is then secreted through TMEM16A and CFTR Cl − channels. Bicarbonate is accumulated inside the cell by means of basolateral transporters and by conversion from CO 2 . Pendrin then mediates the exchange of extracellular Cl − with intracellular HCO 3 − . The apical membrane also contains the ATP12A K + /H + pump and possibly a K + channel. Secretion of K + could be the mechanism controlling the acidification of apical fluid by ATP12A.

Article Snippet: The following primary antibodies and dilutions were used: rabbit monoclonal anti-TMEM16A [SP31] (ab64085, Abcam) at 1:200, mouse IgG1 anti-CFTR (ab570, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics) at 1:250, mouse polyclonal anti-SLC26A4 (H00005172-A01, Abnova) at 1:200, rabbit polyclonal anti-SLC12A2 (HPA020130, Sigma-Aldrich) at 1:1000, rabbit monoclonal anti-CA2 [EPR5195] (ab124687, Abcam) at 1:500, mouse IgG1 anti-MUC5AC (NCL-HGM-45M1, Novocastra) at 1:100, mouse IgG2B anti-acetylated tubulin (7451, Sigma Aldrich) at 1:300, rabbit polyclonal anti-ATP12A (HPA039526, Sigma-Aldrich) at 1:400.

Techniques: