cx45  (Alomone Labs)

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Inhibiting Cx45CT dimerization impairs gap junction intercellular communication Triton X-100 solubility was used to compare MDCK cells expressing Cx45 WT or 6E. A) Western blot image of Triton X-100 extracted protein; total lysate (T), soluble (S), and insoluble (I) fractions. Below is the quantification of protein in the T, S, and I fractions. Statistics represent one-way ANOVA with a Neuman-Keuls post-hoc correction (n=3, ** P

Article Snippet: Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C.

Techniques: Solubility, Expressing, Western Blot

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Inhibiting Cx45CT dimerization alters phosphorylation and protein partner interactions MDCK cells stably expressing Cx45 WT or 6E were cultured, lysed, and immunoprecipitated to assess total phosphorylation levels and interactions with β-tubulin, Drebrin, and Nedd4. A) Representative Western blots of immunoprecipitated Cx45 WT or 6E blotting with a general anti-pY antibody. (n=3, ** P

Article Snippet: Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C.

Techniques: Stable Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Inhibiting Cx45CT dimerization decreases protein degradation Three different MDCK cells expressing either Cx45 WT or 6E were assessed for expression at the protein and transcript levels. A) Western blots and quantification of Cx45 WT, Cx45 6E, and Cx43 expression. B) RT-PCR gel images and quantification of Cx45 WT and 6E transcripts. C) Western blots and quantification of Cx45 WT or 6E subjected to a 12 hr cycloheximide chase. Results presented as the mean + s.e.m. (n=3). Statistics are used to analyze the data were two-tailed unpaired Student’s T-test (A, n=3, * P

Article Snippet: Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Inhibiting Cx45CT dimerization alters Cx45 cellular localization Representative immunofluorescence images (red) of methanol fixed cells stably expressing Cx45 WT (left) or 6E (right). Co-immunostaining (green) for either E-Cadherin (MDCK) or β-catenin (Hek-293T) was used to mark the plasma membranes of the fixed cells (blue = DAPI). A) Clonally selected MDCK cells stably expressing mouse Cx45 WT or 6E. B) Bulk selected MDCK cells stably expressing human Cx45 WT or 6E. C) Bulk selected Hek-293T cells stably expressing mouse Cx45 WT and 6E. D) Isolated time-lapse frames of clonally selected MDCK cells expressing C-terminal eGFP fusions of mouse Cx45 WT or 6E (green). White arrows in the insets indicate gap junction plaques. Scale bar A–C = 20 μm and D = 10 μm.

Article Snippet: Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Immunostaining, Isolation

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Inhibiting Cx45CT dimerization affects hemichannel dye uptake MDCK cells stably expressing Cx45 WT or 6E were subjected to conditions that activate hemichannels activity (Ca 2+ deprivation) and the level of neurobiotin uptake was measured. Representative fluorescent micrographs of cells loaded with neurobiotin and probed with streptavidin-647 conjugate (red). Quantification of DAPI (blue) normalized mean fluorescent intensity (MFI) from 3 replicates, 15 random 100X FOVs were acquired per replicate. Statistics are one-way ANOVA with a Neuman-Keuls multiple comparison test (*** P

Article Snippet: Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C.

Techniques: Stable Transfection, Expressing, Activity Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Sequence alignment of the Cx45CT dimerization domain (A333-N361) from different species Sequence alignment of the A333-N361 region using Clustal Omega to all available Cx45 RefSeq sequences revealed a high degree of conservation within this region, with 72.4% identical, 20.6% conserved, and 6.9% non-conserved residues. For clarity, the sites of point mutations in the Cx45 6E construct are indicated by the single letter code above the alignments. Degree of site conservation (excluding Cx45 6E mutations) is indicated at the bottom, * = identical, : and . = high conservation and intermediate conservation, respectively.

Article Snippet: Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C.

Techniques: Sequencing, Construct

Journal: Cerebral Cortex (New York, NY)

Article Title: Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex—Mouse Subplate Zone

doi: 10.1093/cercor/bhy205

Figure Lengend Snippet: Connexin 26 and 45 immunolabeling. ( A ) P2 mouse. Composite image (Zeiss Mosaic) comprised of multiple tiles under 20× objective. Double immunolabeling of Cx26 (red) and NeuN (green) in P2 animals, showing weak expression of Cx26 in SP neurons at this age. ( B ) 100× objective. Colocalization of NeuN (green) and Cx26 (red) in the SP neurons at P2 age. ( C ) P2 mouse. Composite image comprised of multiple tiles under 20X objective. Double immunolabeling with Cx45 (red) and NeuN (green) showing expression of Cx45 in all cortical layers and the SP zone at P2 age. ( D ) 100× objective. Colocalization of NeuN (green) and Cx45 (red) in the SP neurons at P2 age. ( E ) P10 mouse. Composite image comprised of multiple tiles under a 20× objective. At P10 age, Cx26 (red) expression increases in layers 2, 3, 5 and in SP zone, compared with age P2. ( F ) Images obtained with 100× lens show colocalization of NeuN (green) and Cx26 (red) in the SP neurons at P10. ( G ) P10 mouse. Composite image comprised of multiple tiles under 20× objective. At P10, Cx45 (red) expression increases throughout the cortex and SP zone, compared with P2 mice. ( H ) 100× objective. Colocalization of NeuN (green) and Cx45 (red) in the SP neurons, at P10. Cx45 also expresses in non-neuronal cells of the white matter (arrow). Scale bars in A, C, E, and G = 100 μm; in B, D, F, and H = 20 μm. SP = subplate; WM = white matter. Str. = Striatum; Hoechst nuclei stain (blue).

Article Snippet: Rabbit polyclonal antibodies for Cx26, Cx36, and Cx45 were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Immunolabeling, Expressing, Mouse Assay, Staining