Journal: International Journal of Biological Sciences

Article Title: Hypoxic pancreatic cancer derived exosomal miR-30b-5p promotes tumor angiogenesis by inhibiting GJA1 expression

doi: 10.7150/ijbs.67675

Figure Lengend Snippet: MiR-30b-5p directly regulated GJA1. (A) The prediction of target genes of miR-30b-5p using multiple miRNA databases. Upset plot showing the common target genes in top right box. (B) Venn plot showing the common downregulated genes in HUVEC after exosomes treatment. (C) Heatmap showing the expression of cell junction proteins according to RNA-seq. Red box and green box represented high expression and low expression respectively. (D-G) RT-qPCR verified the expression of selected cell junction proteins in HUVEC after the overexpression of miR-30b-5p. (H) Western blotting verified the expression of GJA1 after the overexpression of miR-30b-5p. (I) The correlation analysis between has-miR-30b-5p and GJA1 based on TCGA_PAAD cohort. (J) The dual-luciferase reporter vector design for the 3' UTR of GJA1. Red box indicated seed region of miR-30b-5p. (K) Relative luciferase activities across groups were tested in dual-luciferase reporter assay.

Article Snippet: The following primary antibodies were used: CD9 (SBI, #EXOAB-CD9A-1, 1:1000, USA), CD63 (SBI, #EXOAB-CD63A-1, 1:1000, USA), HSP70 (SBI, #EXOAB-Hsp70A-1, 1:1000, USA), TSG101 (Proteintech, #14497-1-AP, 1:500, USA), Alix (Proteintech, # 12422-1-AP, 1:500, USA), HIF-1α (CST, # 36169S, 1:1000, USA), β-Actin (Proteintech, #66009-1-Ig, 1:5000, USA), GAPDH (Proteintech, #60004-1-Ig, 1:5000, USA), GJA1 (CST, #83649S, 1:1000, USA).

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Over Expression, Western Blot, Luciferase, Plasmid Preparation, Reporter Assay

Journal: International Journal of Biological Sciences

Article Title: Hypoxic pancreatic cancer derived exosomal miR-30b-5p promotes tumor angiogenesis by inhibiting GJA1 expression

doi: 10.7150/ijbs.67675

Figure Lengend Snippet: Exosomal miR-30b-5p promoted angiogenesis through suppressing GJA1. (A-B) Rescue experiments, the expression of GJA1 in RNA (A) and protein (B) level was detected after co-incubation of hypoxic BxPC-3 derived exosomes and miR-30b-5p inhibitor. (C-D) Rescue experiments, the tube formation and endothelial cell migration were tested after co-transfection of miR-30b-5p mimic and overexpression vector of GJA1. (E) The relative expression of total miR-30b-5p in plasma between PDAC and healthy subjects. (F) The correlation for expression of total miR-30b-5p between peripheral blood plasma and portal vein plasma. (G) ROC plot showing the diagnostic value of total miR-30b-5p in plasma to distinguish PDAC and healthy subjects. (H) The relative expression of exosomal miR-30b-5p in plasma between PDAC and healthy subjects. (I) The correlation for expression of exosomal miR-30b-5p between peripheral blood plasma and portal vein plasma. (J) ROC plot showing the diagnostic value of exosomal miR-30b-5p in plasma to distinguish PDAC and healthy subjects.

Article Snippet: The following primary antibodies were used: CD9 (SBI, #EXOAB-CD9A-1, 1:1000, USA), CD63 (SBI, #EXOAB-CD63A-1, 1:1000, USA), HSP70 (SBI, #EXOAB-Hsp70A-1, 1:1000, USA), TSG101 (Proteintech, #14497-1-AP, 1:500, USA), Alix (Proteintech, # 12422-1-AP, 1:500, USA), HIF-1α (CST, # 36169S, 1:1000, USA), β-Actin (Proteintech, #66009-1-Ig, 1:5000, USA), GAPDH (Proteintech, #60004-1-Ig, 1:5000, USA), GJA1 (CST, #83649S, 1:1000, USA).

Techniques: Expressing, Incubation, Derivative Assay, Migration, Cotransfection, Over Expression, Plasmid Preparation, Diagnostic Assay

Journal: bioRxiv

Article Title: Unbaised intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

doi: 10.1101/2022.08.11.503619

Figure Lengend Snippet: A) HuC/D antibody staining shows that most HuC/D+ cells in the intestine express phox2bb, but also show phox2bb+/HuC/D-cells (progenitors) depicted by arrowheads and phox2bb-;HuC/D+ cells (differentiated neurons) depicted by arrows. Scale bar represents 40 μm. Quantification of the relative amount of double and single positive cells are presented in pie charts (n=9). B) Featureplots showing selective expression of cx43, glula, slc1a2b and s100b in one specific cluster of enteric glia depicted by the circle. C) Live-imaging of 5 dpf tg(8.3phox2bb:kaede);tg(her4:GFP) intestines shows phox2bb-;her4+ cell depicted by the arrowheads that are in close proximity to, or seem to interact with phox2bb+ neurons. Scale bar represents 20 μm.

Article Snippet: Antibody staining using rabbit anti-Cx43 (1:200, Cell Signaling Technologies 83649) was performed as published before ( ).

Techniques: Staining, Expressing, Imaging

Journal: bioRxiv

Article Title: Unbaised intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

doi: 10.1101/2022.08.11.503619

Figure Lengend Snippet: A) Immunohistochemistry staining of Cx43 in the tg(phox2bb:GFP) reporter line shows non overlapping expression in the intestine. Representative images from 4 dpf larvae. Scale bar represents 40 μm. B) Upper graph showing the percentage of larvae that contained Cx43 cells in their proximal, middle and distal intestine. The lower graph shows the number of Cx43 cells per larvae in the proximal, middle and distal intestine (n=19).. B) Pseudotime color-coded featureplot showing a bifurcation towards neuronal differentiation (sensory IPAN: branch1 and inhibitory motor neurons: branch2 containing a secondary branch towards serotonergic neurons marked with the asterix). C) Live-imaging of 5 dpf tg(8.3phox2bb:kaede);tg(her4:GFP) intestines shows phox2bb+;her4+ cells, representing cells undergoing differentiation from progenitor state towards neuronal or glial fate. Scale bar represents 20 μm.

Article Snippet: Antibody staining using rabbit anti-Cx43 (1:200, Cell Signaling Technologies 83649) was performed as published before ( ).

Techniques: Immunohistochemistry, Staining, Expressing, Imaging

Journal: Acta biomaterialia

Article Title: 3D Printed Micro-Scale Force Gauge Arrays to Improve Human Cardiac Tissue Maturation and Enable High Throughput Drug Testing

doi: 10.1016/j.actbio.2018.12.026

Figure Lengend Snippet: Characterization of human iPSC-derived cardiac tissue on force gauge. (A) Bright field images showing remodeling of iPSC-CMs following seeding into the micro-wells over time, scale bar = 500 μm (white) and 100 μm (black), respectively. (B) Immunofluorescence image (left), enlarged Immunofluorescence image (middle) magnifying sarcomere structure, and corresponding bright field image (right) showing tissues stained with alpha-actinin (green), connexin 43 (Red), and nuclei (blue). Scale bar = 50 μm (left and right) and 20 μm (middle), respectively. (C) Plot showing the percentage distribution of sarcomere angles from −90° to +90° respective to pillar bending direction, p<0.0001 for one way ANOVA test, and the percent population in 0° to the force direction is significantly higher than those in all other groups except for the +15° group from Tukey’s post-hoc test. Error bars represent SEM, n = 3 for all data points.

Article Snippet: Samples were then incubated with mouse monoclonal antibodies against alpha-actinin (1:100, Sigma) and rabbit monoclonal antibodies against connexin 43 (1:100, Cell Signaling Technology) overnight at 4 °C.

Techniques: Derivative Assay, Immunofluorescence, Staining

Journal: PLoS Neglected Tropical Diseases

Article Title: Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

doi: 10.1371/journal.pntd.0005830

Figure Lengend Snippet: Reagents for immunofluorescence microscopy.

Article Snippet: connexin 43 mAb , Cell Signaling , 3512S , Hu, Ms, Rt, Nhp , 1:50, Triton.

Techniques: Immunofluorescence, Microscopy

Journal: PLoS Neglected Tropical Diseases

Article Title: Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

doi: 10.1371/journal.pntd.0005830

Figure Lengend Snippet: Overall effect of Leptospira infection on host proteins.

Article Snippet: connexin 43 mAb , Cell Signaling , 3512S , Hu, Ms, Rt, Nhp , 1:50, Triton.

Techniques: Infection

Journal: PLoS Neglected Tropical Diseases

Article Title: Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

doi: 10.1371/journal.pntd.0005830

Figure Lengend Snippet: (A) tight junction protein, zonula occludens-1 (ZO-1) and (B) gap junction protein, connexin 43 (connexin) in HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in a right-hand graph (mean +/- SD, p -value is indicated below each graph, the independent p -value shown as an asterisk for Copenhageni is compared to uninfected and Patoc-infected cells).

Article Snippet: connexin 43 mAb , Cell Signaling , 3512S , Hu, Ms, Rt, Nhp , 1:50, Triton.

Techniques: Staining, Infection

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: ATP release triggered in C6-Cx43 by DF exposure is mediated by hemichannels. (A) Exposure to DF conditions triggered significant ATP release that was blocked by carbenoxolone and the connexin mimetic peptides gap 26 and 27. (B) Botulinum toxin B and bafilomycin A1 had no inhibitory effects; botulinum toxin B even stimulated DF-triggered ATP release. Numbers on the bars indicate n. Star symbols indicate significant differences compared with baseline, whereas the number signs indicate significant differences compared with the corresponding control bar (single symbol, p < 0.05; double symbol, p < 0.01; and triple symbol, p < 0.001). Gap 26 and 27 also significantly depressed baseline ATP release (p < 0.001 and < 0.02 respectively; t test; not indicated on the graph).

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques:

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: (A) Western blots illustrating the absence of P2X7 receptor expression in the various cell lines and conditions used in this study. (B) The influence of different kinases/kinase activators on the phosphorylation status of Cx43 in C6-Cx43 cells. The ratio of nonphosphorylated versus phosphorylated Cx43 is given under each lane, and the ratio for control conditions is converted to 100%. The data are representative for three different experiments. The effect on the phosphorylation status can also be appreciated from the blots shown in Figure 11 (the effect of bFGF is more clear there).

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques: Western Blot, Expressing

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Effect of CT-truncation on LPA and LPS modulation of ATP release in HeLa-Cx43. (A) Experiments on HeLa-Cx43 illustrating inhibition of triggered ATP release by LPA and LPS. Gap 27 blocked the triggered ATP release, whereas botulinum toxin B had no effect (gap 27 also inhibited baseline ATP release, as noted in Figure 1A, p < 0.0001). (B) Experiments on CT-truncated HeLa-Cx43 illustrating absence of triggered ATP release in these cells, no effect of LPA, but significant stimulation of triggered ATP release by LPS. Gap 27 blocked the LPS-enhanced ATP response, but botulinum toxin B had no effect, analogous to the experiments on C6-Cx43 illustrated in Figure 5, C and D. Stars indicate significant differences compared with baseline, whereas the number signs indicate significant differences compared with control.

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques: Inhibition

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Effects of LPS treatment in C6-Cx43. (A) GJ coupling (FRAP) was inhibited by LPS, and this was reversed by PP2 and U0126, not by chelerythrin or PD098.059 (n is number of FRAP experiments on 5 different cultures for control and 2 different cultures for all other conditions). (B) Triggered ATP release was significantly enhanced by LPS, an effect that was reversed by PP2 and U0126, but not by chelerythrin. (C) LPS enhancement of ATP release was strongly inhibited by gap 27, to an extent comparable to that in Figure 1A. (D) Bafilomycin A1 had no effect. Stars indicate significant differences compared with control in A and compared with baseline in B–D; number signs indicate significant differences compared with control. LPS also significantly stimulated baseline ATP release (p < 0.001 in B, <0.05 in C, and <0.001 in D; t test).

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques:

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Western blots illustrating the separation of soluble and insoluble connexin fractions in C6-Cx43, HeLa-Cx43, and HeLa-Cx26 by using Triton X-100 extraction. In HeLa-Cx43, bFGF slightly increased the soluble fraction (at the cost of the insoluble fraction), and a similar tendency was observed for LPS in C6-Cx43. No gross and consistent shifts were however observed over three different experiments.

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques: Western Blot

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Effect of CT-truncation on GJ coupling and ATP release in HeLa-Cx43. (A) Example pictures of FRAP experiments used to investigate dye coupling. The white arrow points to the photobleached cell. (B) Average FRAP recovery traces of the experiments summarized in C. (C) Summary FRAP recovery data determined from data points over the last 25 s. CT truncation had no effect on GJ coupling (n is number of FRAP experiments on 2 different cultures). (D) CT-truncation largely depressed baseline and triggered ATP release. Stars indicate significant differences compared with baseline; number signs indicate significant differences compared with HeLa-Cx43. Baseline in HeLa-Cx43Δ C was significantly below baseline in HeLa-Cx43 (p < 0.0001; t test).

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques:

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Effects of bFGF are analogous to the effects observed with LPS. (A) bFGF significantly inhibited GJ coupling (SLDT) in C6-Cx43, and this effect was partly reversed by genistein (n is number of SLDT images in 4 different cultures). (B) bFGF significantly stimulated the triggered ATP release in C6-Cx43, but this could not be reversed by genistein. (C) bFGF significantly inhibited the triggered ATP release in HeLa-Cx43. (D) bFGF significantly stimulated the triggered ATP release in CT-truncated HeLa-Cx43. Stars indicate significant differences compared with control in A and compared with baseline in B–D. The number signs in B–D indicate significant differences compared with control.

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques:

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Arachidonic acid metabolism and LPS enhancement of ATP release. (A) Inhibition of arachidonic acid production with AACOCF3, of lipo-oxygenase with baicalein, or cyclo-oxygenase with indomethacin all drastically suppressed LPS enhancement of triggered ATP release in C6-Cx43. Similar results were observed in HeLa-Cx43Δ C (B) and HeLa-Cx26 (C). (D–F) Arachidonic acid potentiated DF-triggered ATP release in C6-Cx43, HeLa-Cx43Δ C, and HeLa-Cx26. Stars indicate significant differences compared with baseline; number signs indicate significant differences compared with control.

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques: Inhibition

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Effects of PKC and Src in C6-Cx43. (A) Average FRAP recovery traces (n = 31) in control and after PKC activation with PMA (n = 19). (B) FRAP summary data, showing inhibition of dye coupling by PMA and reversal by chelerythrin or PMA washout (30 min) (n is number of FRAP experiments on 3 different cultures for control and 2 different cultures for all other conditions). (C) Triggered ATP release was inhibited by PMA, an effect that was reversed by chelerythrin. (D) Example pictures illustrating SLDT in control and c-SrcK+-transfected cells. The right border of the scrape is visible as the row of strongly 6-CF–stained cells, and dye spread proceeded from there to the right (bar, 100 μm). A fluorescence intensity profile was derived from each image, and a spatial constant of exponential decay was determined. (E) Summary data of SLDT experiments. Dye coupling was not significantly affected by mock or c-SrcK− transfection, but it was significantly inhibited in c-SrcK+ (n is number of SLDT images obtained in 4 different cultures for control and 2 different cultures for all other conditions). (F) Triggered ATP release was not affected in mock or c-SrcK−, but it was significantly depressed in c-SrcK+. Stars indicate significant differences compared with control in B and E and compared with baseline in C and F. Number signs in C and F indicate significant differences compared with control. PMA significantly depressed baseline ATP release in C (p < 0.001; t test).

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques: Activation Assay, Inhibition, Transfection, Staining, Fluorescence, Derivative Assay

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Effects of LPA treatment in C6-Cx43. (A) GJ coupling (FRAP) was significantly inhibited by LPA, and this was reversed by PP2 and U0126 but not by chelerythrin or PD098.059 (n is number of FRAP experiments on 5 different cultures for control and 2 different cultures for all other conditions). (B) Triggered ATP release was significantly inhibited by LPA, an effect that was reversed by PP2 and U0126, but not by chelerythrin and PD098.059. Stars indicate significant differences compared with control in A and compared with baseline in B. The number signs in B indicate significant differences compared with control.

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques:

Journal:

Article Title: Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

doi: 10.1091/mbc.E06-03-0182

Figure Lengend Snippet: Scheme summarizing the major findings of this study. (A) LPS and bFGF activate various kinases that suppress GJs and hemichannel-mediated ATP release. In addition, both substances also trigger the production of arachidonic acid (AA) via activation of cPLA2 and iPLA2. Arachidonic acid is known to inhibit GJs, and the present study demonstrates that it stimulates hemichannel-mediated ATP release. (B) We conclude that the net effect of LPS and bFGF depends on the balance between activation of kinases, inhibition of hemichannels, and activation of the arachidonic acid metabolic pathway, stimulating hemichannel-mediated ATP release. In HeLa-Cx43 cells the kinase component predominates, whereas in C6-Cx43 it is the arachidonic acid pathway that adds most weight. Truncation of the CT in HeLa-Cx43 removes inhibition by kinases and thereby reveals the arachidonic acid component.

Article Snippet: Blots were probed with a rabbit polyclonal anti-rat Cx43 antibody (1/10,000; Sigma), a rabbit polyclonal anti-rat β-tubulin antibody (1/5000, loading control; Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-rat Cx43 antibody (1/500, epitope located at the intracellular loop; Upstate Cell Signaling, Huissen, The Netherlands), a rabbit polyclonal anti-rat Cx26 (1/2000; Zymed, Invitrogen), or a polyclonal rabbit anti-rat P 2 X 7 antibody (1/1000; Alomone Labs, Jerusalem, Israel) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1/8000-1/4000; Sigma), and detection was done with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Zymed, Invitrogen).

Techniques: Activation Assay, Inhibition