Journal:

Article Title: Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca 2+ and Voltage-Activated Potassium Channels

doi: 10.1124/jpet.109.154104

Figure Lengend Snippet: Expression of BK α and β1 subunits in small mesenteric arteries of SS and BN rats. a, representative images of BK α subunit immunofluorescence staining in SS and BN rats. The expression of α and β1 subunits was assessed by confocal microscopy using selective polyclonal antibodies and the fluorescence-tagged secondary antibody. b, quantification by densitometry demonstrated that expression of α subunit was significantly greater in SS compared with BN rats. Although β1 subunit expression also varied between SS and BN rats, the difference was not significant.

Article Snippet: Thereafter, the vessels were incubated for 1 h at 37°C with the rabbit polyclonal primary antibodies for BK channel α subunit (anti-KCa 2+ 1.1/KCNMA1; Alomone Labs, Jerusalem, Israel; 1:50 dilution in PBS) and β 1 subunit (anti-slob1/KCNMB1; Alomone Labs; 1:100 dilution in PBS).

Techniques: Expressing, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence

Journal: International journal of molecular medicine

Article Title: Secretome of EMSCs neutralizes LPS‑induced acute lung injury via aerosol administration.

doi: 10.3892/ijmm.2023.5307

Figure Lengend Snippet: Figure 1. Morphology and fluorescence identification of EMSCs. (A) EMSCs in P0 and P3. (B) Expression of MSC markers (CD44, Vimentin) and neural crest‑related biomarkers (Cx43, Sox9). EMSCs, ectodermal mesenchymal stem cells; Sox9, SRY‑related high‑mobility group box‑containing protein 9; Cx43, Connexin43.

Article Snippet: Briefly, samples in 24‐well plates were incubated with primary antibodies for CD44 (1:100, Boster, A00052), Cx43 (1:100, Boster, BA1727), Sox9 (1:100, Boster, PA1026‐1), Vimentin (1:100, Boster, PB9359) after being blocked with BSA.

Techniques: Fluorescence, Expressing

Journal: Antioxidants

Article Title: Characterization of the Proprotein Convertase-Mediated Processing of Peroxidasin and Peroxidasin-like Protein

doi: 10.3390/antiox10101565

Figure Lengend Snippet: Generation of a hemagglutinin epitope (HA)-tagged PXDN expressing, knock-in mouse model. Western blot analysis of kidney-, and lung lysates prepared from WT, HA-PXDN knock-in, and PXDN-KO animals. Most upper blots show the presence of the uncleaved and cleaved PXDN with a polyclonal PXDN-specific antibody. The HA signals appear at the expected molecular weight in the knock-in samples; the upper band represents the uncleaved, the lower band indicates the proteolytically cleaved form of PXDN. The lowest blot is the analysis of collagen IV crosslinking in WT, HA-PXDN, and PXDN-KO animals. The collagenase-digested samples were separated with gel electrophoresis, and the membranes were tested for the crosslinked dimeric and uncrosslinked monomeric NC1 domains of α1 isoform of collagen IV. No difference was detected between the wild type and HA-PXDN knock-in mouse in crosslinking activity.

Article Snippet: The digested samples were analyzed with Western blot, and the primary antibody was Anti-Human α1(IV) NC1 clone H11 (Chondrex).

Techniques: Expressing, Knock-In, Western Blot, Molecular Weight, Nucleic Acid Electrophoresis, Activity Assay

Journal: Antioxidants

Article Title: Characterization of the Proprotein Convertase-Mediated Processing of Peroxidasin and Peroxidasin-like Protein

doi: 10.3390/antiox10101565

Figure Lengend Snippet: Characterization of a double-tagged PXDN in PXDN-deficient PFHR-9 cells. ( A ) Analysis of collagen IV crosslinking in WT and KO PFHR-9 cells. The collagenase-digested samples were tested for the crosslinked dimeric and uncrosslinked monomeric NC1 domains of collagen IV α1 isoform. The KO cell line lacks collagen IV crosslinking activity. ( B ) V5 signal is present at the expected molecular weight of PXDN in transfected cells. ( C ) A double FLAG signal appears in transfected cells at the molecular weight of PXDN. The upper band is uncleaved-, the lower one is the cleaved form of the protein. ( D ) Transfection of PXDN-deficient cells with the double-tagged PXDN construct rescues collagen IV crosslinking activity. ( E – H ) Immunofluorescent staining of PXDN-deficient, non-transfected cells (control) shows the lack of FLAG- ( E ) and V5 signal ( F ). TO-PRO-3 (blue) staining ( G ) and the merged picture ( H ) with the nuclear signal proves the presence of the cells. ( I – L ) PXDN-deficient cells transfected with the double-tagged PXDN construct show cell-associated and also network-like extracellular localization (arrows) of the N -terminally FLAG (green) tagged PXDN ( I ). The C-terminal V5 tag signal (red) is cell-associated ( J ). ( K ) TO-PRO-3 signals indicate the presence of nuclei, and on the merged picture, we can observe the partial colocalization of the FLAG- and V5 signals ( L ). The bar indicates 10 µm.

Article Snippet: The digested samples were analyzed with Western blot, and the primary antibody was Anti-Human α1(IV) NC1 clone H11 (Chondrex).

Techniques: Activity Assay, Molecular Weight, Transfection, Construct, Staining

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Downregulation of Cx43 reduces endothelial cell migration. ( a ) Western blot analysis of Cx43, Cx40, and Cx37 revealed that Cx43 is expressed in both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC), whereas Cx40 and Cx37 are not expressed in HMEC. Cx43 (Cx43), Cx40 (Cx), and Cx37 (Cx) expressing HeLa cells were used as the positive controls. CTL: Wildtype HeLa cell lysate. ( b ) Western blot analysis of Cx43 in HMEC transfected with 100 nM siRNA against Cx43 or non-silencing control siRNA (Ctrl) for 48 h. The Cx43 expression was analysed using a polyclonal Cx43 antibody. Immunostaining for GAPDH was used as the loading control. ( c ) Serum-induced cell migration of HMEC transfected with a Cx43-siRNA or a control siRNA (Ctrl) in response to 10% FCS was assessed in a wound assay. Downregulation of Cx43 significantly decreased cell migration, represented as accumulated distance (*** p < 0.001 Ctrl-siRNA vs. Cx43-siRNA, n = 6, 3 independent cell cultures). ( d ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Western Blot, Expressing, Transfection, Control, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Downregulation of Cx43 impairs vessel sprouting ex vivo. ( a ) Downregulation of Cx43 in the mouse aortae was achieved by transfection with Cx43 siRNA (500 nM) or control (Ctrl) siRNA, as assessed by Western blot 48 h later. ( b ) Representative images of aortic sprouts in matrigel. Scale bar represents 200 µm. ( c ) The vessel sprouting area (** p < 0.01 Ctrl-siRNA vs. Cx43-siRNA, n = 2 aortae with eight aortic segments each at 3 days; n = 3–4 aortae with 6–8 aortic segments each at 6 days) and ( d ) the number of sprout bifurcations (** p < 0.01 Ctrl-siRNA vs. Cx43-siRNA, n = 2 aortae with 8 aortic segments each at 3 days; *** p < 0.001 Ctrl-siRNA vs. Cx43-siRNA n = 3–4 aortae with 6–8 aortic segments each at 6 days) were significantly reduced upon downregulation of Cx43, as assessed 3 days and 6 days after transfection in a matrigel assay.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Ex Vivo, Transfection, Control, Western Blot, Matrigel Assay

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Interaction of Cx43 and SHP-2 induces SHP-2 phosphatase activity. ( a ) Upon immunoprecipitation (IP) of SHP-2 from HeLa cells expressing only Cx43, Cx43 was co-immunoprecipitated. As a control, cells transfected with an empty vector (control (CTL)) were used. L: Whole cell lysates. ( b ) SHP-2 was detected upon immunoblotting after immunoprecipitation of Cx43 from HeLa cells expressing only Cx43 in contrast to the control cells (CTL). L: Whole cell lysates. Images originates from the same blot, which was cropped; ( c ) SHP-2 was immunoprecipitated together with Cx43 in HMEC; ( d ) Co-localization of SHP-2 and Cx43 was detected by immunofluorescent staining of HMEC followed by confocal microscopic imaging. Scale bar represents 20 µm; ( e ) SHP-2 phosphatase activity was significantly increased in HeLa cells expressing Cx43 compared to HeLa cells transfected with an empty vector (* p < 0.05, n = 4 independent cell cultures), as assessed by detection of dephosphorylation of a phosphate analogue in SHP-2 immunoprecipitates.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, Immunoprecipitation, Expressing, Control, Transfection, Plasmid Preparation, Western Blot, Staining, Imaging, De-Phosphorylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Inactivation of SHP-2 reduces endothelial cell migration. ( a ) Overexpression of a dominant negative substrate trapping mutant SHP-2 (SHP-2 CS) in HMEC significantly reduced serum (10%) induced endothelial cell migration, as assessed by the accumulated distance in µm in an in vitro wound assay (* p < 0.05, n = 3 independent cell cultures) compared to SHP-2 WT. Additional knock-down of Cx43 (Cx43 siRNA) did not further decrease migration (ns: not significant, n = 3 independent cell cultures). ( b ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Over Expression, Dominant Negative Mutation, Mutagenesis, In Vitro, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: The Cx43 and SHP-2 interaction is vital for endothelial migration. ( a ) Treatment of HMEC overexpressing a constitutively active mutant SHP-2 (SHP-2 EA) with Cx43 siRNA did not rescue the reduced migratory response caused by Cx43 knock-down (Cx43 siRNA) (ns: not significant, n = 3 independent cell cultures) compared to SHP-2 EA cells treated with control siRNA (Ctrl) (* p < 0.05, n = 3 independent cell cultures), as assessed by the accumulated distance in µm in an in vitro wound assay (* p < 0.05, n = 3 independent cell cultures). ( b ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Mutagenesis, Knockdown, Control, In Vitro

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 3 Immunohistochemical analysis of the distinct presence of Cx43 and BMP-15 in the oophorons of each group (× 200) A-F: Cx43; G-L: BMP-15. A, G: control group; B, H: model group; C, I: positive group; D, J: low dose of BSJPP group; E, K: moderate dose of BSJPP group; F, L: high dose of BSJPP group. Positive group treated with premarin (0.03 mg/kg) once daily for 90 d. Con- trol and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Immunohistochemi- cal analysis clearly revealed the presence of Cx43 and BMP-15 in the oophorons of control mice and those treated with BSJPP (D, E) and premarin compared with the model group. BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Immunohistochemical staining, Control, In Vivo

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 4 Expression Cx43 and BMP-15 mRNA in the ovaries of mice in each group. 1: control group; 2: model group; 3: positive group; 4: low dose of BSJPP group; 5: moderate dose of BSJPP group; 6: high dose of BSJPP group. Positive group treated with pre- marin (0.03 mg/kg) once daily for 90 d. Control and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Compared with the model group, significantly upregulated expression of both Cx43 and BMP-15 was detected in the control group and the groups treated with BSJPP (M and L groups). BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Expressing, Control, In Vivo