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: Physiological Reports

Article Title: Western blot analysis of BK channel β 1‐subunit expression should be interpreted cautiously when using commercially available antibodies

doi: 10.14814/phy2.12189

Figure Lengend Snippet: Representative western blot obtained using anti‐BK β 1‐subunit antibodies in MA, colonic, or kidney tissues from WT and BK β 1‐KO mice, (A) Alomone Labs (APC‐036), (B) Pierce (PA5‐28284), and (C) Abcam (Ab3587) antibodies detected a protein band at ~28 kDa or ~38 kDa in colons or MA from both mice. The bands in Abcam sets were diminished after preincubation of the primary antibody with the competing peptide. (D) Pierce (PA1‐924), (E) Santa Cruz (sc‐14749), and (F) Biorbyt (orb‐101774) antibodies did not detect any band at ~28 kDa or ~21 kDa in MA or colons from WT mice. (G) Alomone Labs (APC‐036), Pierce (PA1‐924), Abcam (Ab3587), and Biorbyt (Orb‐101774) antibodies did not detect protein band at ~28 kDa, ~38 kDa, or ~21 kDa in kidneys from WT mice. (H) Pierce (PA5‐28284) and (I) Santa Cruz (sc‐14749) antibodies detected the protein band at ~28 kDa in kidneys from both mice. β ‐actin was reblotted on each membrane after anti‐BK β 1‐subunit antibody was stripped. All representative blot images from kidney are in the tissue from same WT or BK β 1‐KO mouse, and blotted with primary anti‐BK β 1‐subunit antibody at 1:200. Arrows indicate the manufacturer's recommended molecular weight of BK β 1‐subunit protein.

Article Snippet: We also tested an anti‐BK α ‐subunit antibody (APC‐107, anti‐ KCa1.1 , 1:500; Alomone Labs, Jerusalem, Israel) in protein extracts from colons and kidneys, to confirm BK channel expression in these tissues.

Techniques: Western Blot, Molecular Weight

Journal: Physiological Reports

Article Title: Western blot analysis of BK channel β 1‐subunit expression should be interpreted cautiously when using commercially available antibodies

doi: 10.14814/phy2.12189

Figure Lengend Snippet: (A) Representative amplification plots and agarose gel separation of real‐time RT‐PCR analysis of BK β 1‐subunit and GAPDH in MA, colons, and kidneys from WT and BK β 1‐KO mice. The expression threshold was set at 0.22, a level above background fluorescence but within the linear phase of the amplification plot. The intersection between the threshold level and the amplification plot is the Ct value, which correlates with the amount of template in the sample. Ct values over 35 are excluded, as these values approach the sensitivity limits of the Taqman assay. Amplification of real‐time RT‐PCR products was seen at 75 bp in tissues from WT animals only. Con, nontemplate control. (B) Representative western blot obtained using anti‐BK α ‐subunit antibody in colonic and kidney tissues from WT and BK β 1‐KO mice. Antibody detected a protein band at ~100 kDa in all tissues from WT and BK β 1‐KO mice. The signals are blocked by preincubation with the antibody competing peptide (CP). Arrows indicate the manufacturer's recommended molecular weight of BK α ‐subunit protein.

Article Snippet: We also tested an anti‐BK α ‐subunit antibody (APC‐107, anti‐ KCa1.1 , 1:500; Alomone Labs, Jerusalem, Israel) in protein extracts from colons and kidneys, to confirm BK channel expression in these tissues.

Techniques: Amplification, Agarose Gel Electrophoresis, Quantitative RT-PCR, Expressing, Fluorescence, TaqMan Assay, Western Blot, Molecular Weight

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: 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