Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Advanced Glycation End Products Downregulate Connexin 43 and Connexin 40 in Diabetic Atrial Myocytes via the AMPK Pathway

doi: 10.2147/DMSO.S419189

Figure Lengend Snippet: Expression of Cx43 and Cx40 proteins in atrial tissues from Control and Diabetic groups. ( A ) Representative blots of Cx43 and Cx40 proteins in atrium of diabetic rat (n=6). ( B and C ) Densitometry analysis of Cx43 and Cx40 proteins in atrium of diabetic rat (n=6). ( D ) Representative histological fixed sections of atrial tissues stained with Immunohistochemistry. * p <0.05.

Article Snippet: After blocking with 5% non-fat milk in tris-buffered saline containing Triton-X100, membranes were hybridized overnight with the specific primary antibodies AGE (1:1000, ab23722, Abcam), rabbit polyclonal to RAGE (1:1000, ab65965, Abcam), p-AMPK (1:1000, 2535S, Cell Signaling Technology), AMPK (1:1000, 2532S, Cell Signaling Technology), Cx43 (1:500, sc-365107, Santa Cruz Biotechnology, Dallas, Texas, USA) and Cx40 (1:500, sc-271837, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Staining, Immunohistochemistry

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Advanced Glycation End Products Downregulate Connexin 43 and Connexin 40 in Diabetic Atrial Myocytes via the AMPK Pathway

doi: 10.2147/DMSO.S419189

Figure Lengend Snippet: Altered protein expression levels of Cx43 and Cx40 in HL-1 cells and primary atrial myocytes treated with AGEs. ( A ) Representative blots of Cx43 and Cx40 proteins in each HL-1 cell group (n=6). ( B and C ) Densitometry analysis of Cx43 and Cx40 proteins in each HL-1 cell group (n=6). ( D ) Representative blots of Cx43 and Cx40 proteins in each primary rat atrial myocyte group (n=6). ( E and F ) Densitometry analysis of Cx43 and Cx40 proteins in each primary rat atrial myocyte group. * p <0.05.

Article Snippet: After blocking with 5% non-fat milk in tris-buffered saline containing Triton-X100, membranes were hybridized overnight with the specific primary antibodies AGE (1:1000, ab23722, Abcam), rabbit polyclonal to RAGE (1:1000, ab65965, Abcam), p-AMPK (1:1000, 2535S, Cell Signaling Technology), AMPK (1:1000, 2532S, Cell Signaling Technology), Cx43 (1:500, sc-365107, Santa Cruz Biotechnology, Dallas, Texas, USA) and Cx40 (1:500, sc-271837, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Advanced Glycation End Products Downregulate Connexin 43 and Connexin 40 in Diabetic Atrial Myocytes via the AMPK Pathway

doi: 10.2147/DMSO.S419189

Figure Lengend Snippet: Effects of AMPK activation on AGEs-induced Cx43 and Cx40 alteration in atrial cells. ( A ) Representative blots of p-AMPK, AMPK, Cx43 and Cx40 proteins in each cell group (n=6-9). ( B-E ) Densitometry analysis of p-AMPK, AMPK, Cx43 and Cx40 proteins in each cell group (n=6-9). * p <0.05, # p >0.05.

Article Snippet: After blocking with 5% non-fat milk in tris-buffered saline containing Triton-X100, membranes were hybridized overnight with the specific primary antibodies AGE (1:1000, ab23722, Abcam), rabbit polyclonal to RAGE (1:1000, ab65965, Abcam), p-AMPK (1:1000, 2535S, Cell Signaling Technology), AMPK (1:1000, 2532S, Cell Signaling Technology), Cx43 (1:500, sc-365107, Santa Cruz Biotechnology, Dallas, Texas, USA) and Cx40 (1:500, sc-271837, Santa Cruz Biotechnology).

Techniques: Activation Assay

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Advanced Glycation End Products Downregulate Connexin 43 and Connexin 40 in Diabetic Atrial Myocytes via the AMPK Pathway

doi: 10.2147/DMSO.S419189

Figure Lengend Snippet: Decreased expression of Cx43 and Cx40 by downregulation of AMPK. ( A ) Representative blots of AMPK, Cx43 and Cx40 proteins in HL-1 cells transfected with AMPK α1 siRNA (n=6-9). ( B-D ) Representative densitometry analysis of AMPK, Cx43 and Cx40 proteins in HL-1 cells transfected with AMPK α1 siRNA (n=6-9). ( E ) Representative blots of AMPK, Cx43 and Cx40 proteins in HL-1 cells transfected with AMPK α2 siRNA (n=6-9). ( F-H ) Representative densitometry analysis of AMPK, Cx43 and Cx40 proteins in HL-1 cells transfected with AMPK α2 siRNA (n=6-9). * p <0.05.

Article Snippet: After blocking with 5% non-fat milk in tris-buffered saline containing Triton-X100, membranes were hybridized overnight with the specific primary antibodies AGE (1:1000, ab23722, Abcam), rabbit polyclonal to RAGE (1:1000, ab65965, Abcam), p-AMPK (1:1000, 2535S, Cell Signaling Technology), AMPK (1:1000, 2532S, Cell Signaling Technology), Cx43 (1:500, sc-365107, Santa Cruz Biotechnology, Dallas, Texas, USA) and Cx40 (1:500, sc-271837, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 2. Antibodies against Cx40 specifically recognize gap junctions. (A) The antibodies to Cx40 that were used throughout this study did not stain the internal elastic lamina and smooth muscle cells (SMC) of a rat aorta. In contrast, the protein A-gold particles that were reacted with the antibodies selectively decorated minute areas of apposition (arrows) between the membranes of adjacent endothe- lial cells (EC). (B) At higher magnification, the area of membrane apposition pointed by the arrows in (A) features the pentalaminar appearance characteristic of a gap junction plaque and is decorated by 15 nm gold parti- cles, reflecting the presence of Cx40. (C) Im- munofluorescence labeling with the same anti- bodies located Cx40 at discrete spots along the endothelial layer of the rat aortic wall but not over the media. Note that the endothelial labeling was interrupted at sites of cell nuclei (arrow heads). (D) is the phase contrast view of the field shown in (C). Bar represents 3 m in (A), 1.1 m in (B), and 70 m in (C and D). L, lumen of vessel.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Staining, Membrane, Labeling

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 3. Distribution of Cx40 mRNA in the kidney. In situ hybridization using two differ- ent antisense rat cDNA probes specific for Cx40 demonstrated that the mRNA coding for this protein is expressed in the endothelium of small kidney vessels (A), including juxtaglom- erular arterioles (arrow heads in C and D) and glomerular capillaries (C and D). No sig- nal was detected when sections of the very same kidney were hybridized with a sense probe (B). Bar represents 100 m.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: In Situ Hybridization

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 4. Cx40 and Cx43 are differentially dis- tributed in the kidney. Specific antibodies against the carboxy terminus of Cx40 showed abundant levels of this protein along the endo- thelium of small kidney vessels (A) and be- tween media cells of the afferent arteriole (ar- rowhead in B and C). Much less Cx40 was also seen between the mesangial and endothe- lial cells of glomeruli (B). (C) is the phase contrast of the field shown in (B). Antibodies to Cx43 showed the presence of this connexin between the endothelial cells of medium size (D) and small kidney arterioles, including the afferent arteriole (arrows in E). In contrast, Cx43 was not found between the media cells of the latter vessel (arrowhead in E) and was barely visible within glomeruli (E). (F) The phase contrast view of the field shown in (E). The bar represents 15 m.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques:

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 5. Cx40 is expressed in the portion of the afferent arteriole that comprises renin-secreting cells. Immunolabeling of the cortex of a rat kidney showed that Cx40, as revealed using a fluorescein-tagged secondary antibody, was abundant in the juxtaglomerular portion of the afferent arteriole (A), which was also positive for renin-containing cells, as revealed using a rhodamin-tagged secondary antibody (B). Double exposure of the same area showed that most of the Cx40 signal colocalized with that of renin, resulting in a yellow staining of the juxtaglomerular portion of the afferent arteriole (C). Under similar conditions, the antibody to Cx40 usually elicited a more extensive staining in the afferent arteriole of hypertensive (E) than normotensive rats (D). The bar represents 22 m in (A–C) and 11 m in (D and E).

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Immunolabeling, Staining

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 6. Cx40 links renin-containing cells, en- dothelial cells, as well as renin and endothelial cells of the afferent arteriole. The ultrastruc- tural distribution of Cx40 was studied in sec- tions of the juxtaglomerular portion (gc, glo- merular capillary) of afferent arterioles (aa). (A) As judged by the decoration of the protein A-gold particles that were reacted with the anti- bodies to Cx40, this connexin was selectively located at minute areas of apposition between the membranes of renin-containing (rc; rect- angle 1) and endothelial cells (ec; rectangle 2). (B and C) Higher magnification views of the fields outlined by rectangles 1 and 2 in (A). (D) Renin-containing cells were identified by their characteristic secretory granules, which were decorated by protein A-gold particles when reacted with antirenin antibodies. (E) Cx40 was also found at contacts between the membranes of renin-containing and endothe- lial cells (rectangle 3). (F) Higher magnifica- tion view of the field outlined by rectangle 3 in (E). (G and H) when the orientation of sectioning was favorable, the areas immunola- beled for Cx40 between endothelial cells (G) and between renin-containing cells (H) dis- played the pentalaminar appearance of mem- brane appositions (the arrowheads point to the middle dark line), characteristic of gap junctions in aldehyde-fixed tissues. The bar represents 1.5 m in A; 150 nm in B, C, and F; 500 nm in D and E; 125 nm in G; and 112 nm in H.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques:

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 7. Expression of Cx40, Cx43, and renin genes in kidneys of normotensive and hyper- tensive rats. (A and B) Northern blots re- vealed that compared with the levels normal- ized to the GAPDH signal observed in sham- operated animals, the levels of the Cx40 tran- script were increased in both left (LK) and right (RK) kidneys of 2K1C rats, whereas those of the Cx43 transcript were increased only in the unclipped right kidney (RK). The blots also show that the expression of the renin gene was markedly enhanced in the clipped left kidney (LK) and decreased in the unclipped right kidney of the hypertensive 2K1C rats. In the very same samples, levels of GAPDH mRNA were not modified. In left panels, each lane shows a sample of left kidney from a different rat. In right panels, corresponding lanes show samples of right kidneys of the very same animals. All lanes were loaded with 6 g poly A mRNA. (C) Quantitative assess- ment of nine experiments (one per rat) con- firmed that the levels of Cx40 mRNA were increased (P 0.01) approximately twofold over control values in both left (LK) and right (RK) of 2K1C rats. In contrast, those of Cx43 were significantly increased only in the right unclipped kidney of 2K1C rats. Values repre- sent ratios of densitometric measurements of either Cx40 or Cx43 and GAPDH mRNAs and are expressed as mean SEM. *P 0.05; **P 0.01 level.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Expressing, Northern Blot, Control

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 8. Expression of the Cx40 gene in aortic endothelial cells of 2K1C hypertensive rats. (A) Northern blots revealed that the levels of Cx40 mRNA were higher in the aorta of hypertensive 2K1C rats than in that of sham-operated, normotensive controls. Each lane is from a different animal and was loaded with 15 g RNA. (B) Quantitative densitometric analysis of eight independent experiments (one per rat) showed that relative to the levels of GAPDH mRNA that were constant, those of Cx40 mRNA were twice as high in 2K1C than in sham-operated rats. *P 0.01.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Expressing, Northern Blot