rabbit anti-cx43 polyclonal primary antibody  (Santa Cruz Biotechnology)

Journal: Genetics and molecular research : GMR

Article Title: Tiaogan Qingxin Granule treatment increases myocardial connexin 43 expression in a rat model of arrhythmia.

doi: 10.4238/gmr.15027934

Figure Lengend Snippet: Figure 2. Western blots of Cx43 and GAPDH proteins in each group. Lane 1, Sham-operated group. Lane 2, Arrhythmia model group. Lane 3, Wenxin Granule group. Lane 4, Tiaogan Qingxin Granule group.

Article Snippet: The proteins were diluted with 0.9% NaCl to obtain same concentration and same amounts were denatured at 100°C for 3 min. Proteins were then separated by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, stored at 4°C for 80 min, and blocked with 5% TBST in skim milk for 1 h. Subsequently, the membranes were treated with rabbit anti-mouse Cx43 polyclonal primary antibody (diluted 1:1,000 in TBST; Cell Signaling Technology, Danvers, MA, US) at 4°C overnight, washed, and incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:2,000; ZSGB-BIO, Beijing, China) for 1 h at room temperature.

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation

doi: 10.3892/mmr.2015.4193

Figure Lengend Snippet: Immunofluorescence and confocal laser scanning microscopy of Cx43 and p-Cx43. High-intensity specific immunoreactive signals of Cx43 and p-Cx43 were clearly identified and regularly distributed in ZP123 groups compared to the control groups. With the duration of VF, Cx43 and p-Cx43 signals decreased and distributed in heterogeneity. Magnification, ×400. p-Cx43, phosphorylated Cx43; ZP123, rotigaptide; VF, ventricular fibrillation; NS, normal saline; Cx, connexin.

Article Snippet: The polyclonal rabbit anti-dog primary antibodies for Cx43 (AB1727; 1:100 dilution in PBS; EMD Millipore) and p-Cx43 (P3859Rb-p; 1:100 dilution in PBS; USCN Life Science, Inc., Wuhan, China) detection were added and slides were incubated overnight at 4°C.

Techniques: Immunofluorescence, Confocal Laser Scanning Microscopy, Control, Saline

Journal: Molecular Medicine Reports

Article Title: Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation

doi: 10.3892/mmr.2015.4193

Figure Lengend Snippet: Western blot analysis of Cx43, p-Cx43 and β-actin. Values are expressed as the mean ± standard error of the mean. * P<0.05 vs. sham control; 2 P<0.05 vs. the corresponding control NS group. β-actin was used as loading control. p-Cx43, phosphorylated Cx43; ZP123, rotigaptide; VF, ventricular fibrillation; NS, normal saline; Cx, connexin.

Article Snippet: The polyclonal rabbit anti-dog primary antibodies for Cx43 (AB1727; 1:100 dilution in PBS; EMD Millipore) and p-Cx43 (P3859Rb-p; 1:100 dilution in PBS; USCN Life Science, Inc., Wuhan, China) detection were added and slides were incubated overnight at 4°C.

Techniques: Western Blot, Control, Saline

Journal: Molecular Medicine Reports

Article Title: Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation

doi: 10.3892/mmr.2015.4193

Figure Lengend Snippet: Correlation between Cx43 expression and defibrillation success rate. Cx, connexin.

Article Snippet: The polyclonal rabbit anti-dog primary antibodies for Cx43 (AB1727; 1:100 dilution in PBS; EMD Millipore) and p-Cx43 (P3859Rb-p; 1:100 dilution in PBS; USCN Life Science, Inc., Wuhan, China) detection were added and slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation

doi: 10.3892/mmr.2015.4193

Figure Lengend Snippet: Correlation between the expression of Cx43 and the average defibrillation energy. Cx, connexin.

Article Snippet: The polyclonal rabbit anti-dog primary antibodies for Cx43 (AB1727; 1:100 dilution in PBS; EMD Millipore) and p-Cx43 (P3859Rb-p; 1:100 dilution in PBS; USCN Life Science, Inc., Wuhan, China) detection were added and slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation

doi: 10.3892/mmr.2015.4193

Figure Lengend Snippet: Correlation between the expression of p-Cx43 and the success rate of first three defibrillations. p-Cx43, phosphorylated connexin43.

Article Snippet: The polyclonal rabbit anti-dog primary antibodies for Cx43 (AB1727; 1:100 dilution in PBS; EMD Millipore) and p-Cx43 (P3859Rb-p; 1:100 dilution in PBS; USCN Life Science, Inc., Wuhan, China) detection were added and slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 1: Cx43 mutants that interfere with AP-2/clathrin binding. (A) The Cx43 C-terminus harbors three canonical tyrosine-based sorting signals of the type YXXΦ at positions Y230VFF, Y265AYF, and Y286KLV, which we named S1, S2, and S3 (shown in red), that can function as putative binding sites for the classical PM clathrin adaptor, AP-2. To test their role in recruiting AP-2 to Cx43, we generated a set of mutants that abolished the AP-2 binding capacity by mutating known critical residues of the motifs (Y or F) into H or A, or by deleting the amino acid residues of the motifs, either partially (three amino acid residue deletions) or entirely (seven or more amino acid deletions) (depicted with asterisks, see the text for details). Single-, double-, and triple-site mutants (depicted S1, S2, S3, S2+3, and S1+2+3) were constructed. Mutants 2–10 characterized in this work are shown. (B) Immunoblot analysis (using anti-GFP antibodies) of wild type (lane 1) and mutant Cx43-GFP constructs (lanes 2–10). Wild type and mutants produced Cx43 proteins that migrated on SDS–PAGE gels corresponding to their predicted molecular weight. M, marker proteins.

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Binding Assay, Generated, Residue, Construct, Western Blot, Mutagenesis, Produced, SDS Page, Molecular Weight, Marker

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 2: Cx43 mutants with AP-2 binding site S2 or S3 mutated coprecipitate reduced amounts, while mutants with both (S2+3) or all three (S1+2+3) sites mutated no longer coprecipitate clathrin and clathrin accessory proteins. HeLa cells were transfected with GFP-tagged (A) and untagged (B) Cx43 wild type (lane 1); single F268A (S2), Y286H (S3) (lanes 2 and 3); double F268A+Y286H (S2+3), ΔY265AY+ΔP283PGYKLV (S2+3) (lanes 4 and 5); and triple Y230H+F268A+Y286H (S1+2+3), Y230H+ΔY265AY+ΔP283PGYKLV (S1+2+3) (lanes 6 and 7) mutants. Cells were lysed in SDS-free 1× RIPA buffer, and protein complexes were immunoprecipitated using anti-GFP (A) or anti-Cx43 (B) magnetic beads. Bound and coprecipitated endocytic proteins were detected by Western blot analyses using antibodies specific for GFP (row 1 in A), Cx43 (row 1 in B), α-adaptin (AP-2, row 2), Dab2 (row 3), clathrin heavy chain (CHC, row 4), and α-tubulin (loading control, row 5). Cx43 wild type and single-site mutants S2 and S3 coprecipitated AP-2, Dab2, and clathrin, while double (S2+3) and triple (S1+2+3) mutants did not. HeLa cell lysates and beads only were analyzed in control (lanes 8 and 9). Note that both single mutants S2 and S3 precipitated approximately half the amounts of clathrin and clathrin adaptors compared with wild-type Cx43 (see quantitative graphed analyses on the right). Representative blots are shown on the left, and averaged data of three independent experiments are shown on the right. Note that increasing amounts of Cx43 proteins were immunoprecipitated, correlating with the increasing stability of the mutant proteins (see Figure 6) and that a small amount of endogenous Cx43 was detected in overexposed membranes (row 1, lane 9 in B; quantified in Figure S1). Significant differences (p < 0.05) are shown in this and following figures. Results corroborate that motif S1 is not a functional AP-2 binding site.

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Control, Mutagenesis, Functional Assay

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 3: The accessory clathrin adaptor, Dab2, interacts indirectly with Cx43 via AP-2. (A) HeLa cells were transfected with wild-type Cx43-GFP (a), Cx43-ΔL254–290-GFP (b, mutant S2+3), and Cx43-ΔL254–CT-GFP (c, mutant S2+3), and GJ plaques and AGJ vesicles were detected by their emitted fluorescence (green). In-cell protein colocalization assays between Cx43 and Dab2 were performed with mouse anti-Dab2 and rabbit anti-Cx43 antibodies using in situ Duolink technology (red fluorescence). Cx43 wild-type AGJ vesicles colocalized with Dab2, detectable as red/yellow puncta in the magnified views (a). The Cx43-ΔL254–290 mutant is unable to recruit AP-2 and also does not recruit Dab2, as indicated by the negative Duolink signal (b). The Cx43-ΔL254–CT mutant is also unable to interact with the Cx43 antibodies and served as a negative Duolink control (c). Representative images are shown. (B) Quantitative analyses of results described in (A) revealing the impaired ability of AP-2 binding–deficient Cx43 mutants to recruit Dab2. Results are consistent with coimmunoprecipitation results presented in Figure 2.

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Transfection, Mutagenesis, Fluorescence, In Situ, Control, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 4: AP-2 binding–deficient Cx43 mutants have more GJ channels in their apposed PMs. (A) Untagged wild type and S2, S3, and S2+3 Cx43 mutants were cotransfected with mCherry-H2B (red; to better reveal transfected cell pairs) into HeLa cells. Cx43 was stained for immunofluorescence analyses using primary rabbit anti-Cx43 and secondary goat anti-rabbit Alexa Fluor 488 (green) labeled antibodies. Images of transfected cell pairs were acquired using a 60× oil-immersion objective. Three representative images of wild type (a) and selected mutants (b, F268A [S2]; c, Y286H [S3]; d, F268A+Y286H [S2+3]; e, ΔY265AY+ΔP283PGYKLV [S2+3]) are shown. (B) Quantitative analyses of the number of GJ channels present in apposing PMs (performed by measuring PM localized Alexa Fluor 488 fluorescence intensity of wild type and selected S2, S3, and S2+3 mutants) revealed a twofold increase in Cx43-F268A (S2) and Cx43-Y286H (S3) mutants (blue bars) and a threefold increase in Cx43-F268A+Y286H and ΔY265AY+ΔP283PGYKLV (S2+3) mutants (orange/ red bars) compared with wild-type Cx43–expressing cells (green bar).

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Binding Assay, Transfection, Staining, Immunofluorescence, Labeling, Fluorescence, Expressing

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 5: GJ plaques assembled by AP-2 binding–deficient Cx43 mutants are internalization impaired. (A) HeLa cells were transfected with GFP-tagged wild type, S2, S3, and S2+3 mutant constructs and imaged every 2 or 5 min for up to 72 h (phase-contrast and green fluorescence channels). GJ plaque formation, maturation, and endocytosis were captured (depicted by arrows). Selected merged image frames of wild type, F268A (S2), Y286H (S3), and F268A+Y286H-GFP and ΔL254–290-GFP (S2+3) are shown (see also Movies S1–S5). (B) Quantitative analysis of time periods required for GJ plaques to constitutively turn over (form, mature, and be removed) revealed that GJ plaques assembled of wild type Cx43, on average, turned over in ∼5 h (green bar), while GJ plaques assembled from S2 and S3 mutants turned over in 18–19 h (blue bars), and GJ plaques assembled from S2+3 double mutants were removed after ∼30 h (red bar). (See Table S1 for complete data set.)

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Binding Assay, Transfection, Mutagenesis, Construct, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 6: AP-2 binding–deficient Cx43 mutant proteins exhibit longer half-lives. HeLa cells were transfected with GFP-tagged (A) or untagged (C) wild type, S2, S3, and S2+3 Cx43 mutant constructs and treated with cycloheximide the following day to block protein biosynthesis. Cells were lysed at indicated time points posttreatment and examined by SDS–PAGE and Western blot analyses using anti-GFP (A) and anti-Cx43 (C) antibodies. All membranes were stripped and reprobed with anti–α-tubulin antibodies as a loading control. Representative Western blot analyses for Cx43 wild type (a), F268A (b, S2), Y286H and ΔP283PGYKLV (panels c and d in A, S3), F268A+Y286H and ΔY265AY+ΔP283PGYKLV (panel d in C and panels e and f in A, S2+3) are shown. (B, D) Normalized quantitative analyses of Cx43 protein that remained after the indicated times in three independent analyses indicates that S2 and S3 mutant proteins have approximately doubled half-lives (∼2 h, blue curves), while double S2+3 mutants again have about doubled half-lives (∼4 h, orange/red curves) when compared with wild-type Cx43 protein (estimated half-life of ∼1 h, green curve).

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Binding Assay, Mutagenesis, Transfection, Construct, Blocking Assay, SDS Page, Western Blot, Control

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 7: AP-2 binding–deficient Cx43 mutants become donor cells. (A) HeLa cells were separately transfected with wild-type Cx43-mApple (red) or GFP-tagged wild type, S2, S3, or S2+3 mutant constructs (green) and cocultured. Cell pairs of wild-type Cx43-mApple and mutant/wild-type-GFP–expressing cells assemble heterotypic GJs (yellow in the overlays). Heterotypic AGJs (also appearing yellow) are depicted with arrows. Representative images of wild-type Cx43-GFP (a), F268A-GFP (b, S2), Y286H-GFP (c, S3), and F268A+Y286H-GFP (d, S2+3)–expressing cells paired with wild-type Cx43-mApple– expressing cells of three independent experiments are shown. (B) Heterotypic AGJs were counted, and their cellular location in wild-type Cx43-mApple–expressing cells vs. mutant Cx43-GFP–expressing cells was quantified. Cx43 wild-type/ wild-type cell pairs exhibited an unbiased average ratio of 1:1 (bar 1), while Cx43 mutants (S2, S3, or S2+3, bars 2–4) were progressively inefficient in internalizing their plaques. Significance values between cell-pair groups, and between cell types within groups compared with wild-type/wild-type pairs are shown above and within columns, respectively.

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: Binding Assay, Transfection, Mutagenesis, Construct, Expressing

Journal: Molecular Biology of the Cell

Article Title: Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

doi: 10.1091/mbc.e13-02-0111

Figure Lengend Snippet: FIGURE 8: Model of Cx43 recruiting clathrin and endocytic clathrin adaptors. Top, Cx43 encodes three canonical tyrosine-based sorting motifs of the type “YXXΦ” in its C-terminus (S1, S2, and S3). Sorting signals S2 and S3 both can recruit the classical clathrin adaptor AP-2 to Cx43-GJs that are destined for internalization. Middle, internal ization-destined Cx43 binds the μ2 subunit of AP-2 via its Y265AYF (S2) and/or its Y286KLV (S3) motif. AP-2 then recruits clathrin either directly via its β2 subunit that binds to the terminal domain of CHC, and/or it interacts via its α subunit with clathrin-bound Dab2. Bottom, as a consequence, one Cx43 polypeptide may recruit up to four clathrin molecules, potentially generating the principal molecular structure required for an efficient endocytosis of GJs.

Article Snippet: Cells were blocked with 10% FBS in PBS at room temperature for 1 h. Cells were incubated with rabbit polyclonal anti-Cx43 primary antibodies (cat. no. 3512; Cell Signaling Technology, Danvers, MA) at 1:500 dilution at 4°C overnight.

Techniques: