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. 2001 Dec 24;155(7):1307–1318. doi: 10.1083/jcb.200102017

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Copyright © 2001, The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 6. — GJ assembly following the expression of vector, ^S364PCx43, ^S364ECx43, ^S364ACx43, or ^WTCx43 in WT fibroblasts. (A) Comparison of GJ assembly between WT fibroblasts (10-3) transfected with either ^S364PCx43 or ^S364ECx43 and treated with or without 8Br-cAMP for the entire experiment (*, injected cell). (B) Example of immunoblots of cell homogenates following two different assembly experiments using either WT fibroblasts transiently transfected with ^M257Cx43 or ^S364PCx43. Cells were transfected 48 h prior to experimentation with either vector alone (pIRES; 1, 2, 5, and 6) or mutant construct (3, 4, 7, and 8). Cells were dissociated and recovered for 4 h 15 or 23 min, reaggregated for 15 (^M257Cx43) or 7 (^S364PCx43) min, and treated either without (1 and 3) or with (2 and 4) 8Br-cAMP for the entire GJ assembly experiment. To detect endogenous ^WTCx43 and transfected ^M257Cx43 (arrowhead, first panel) membranes were labeled with NH₂-terminal Cx43 antibody (1:8,000) detected with goat anti–rabbit peroxidase antibody (1:10,000). To detect mutant and ^WTCx43 expression (second panel), membranes were labeled with Cx43 CT antibody (1:8,000; Sigma-Aldrich) detected with goat anti–rabbit peroxidase antibody (1:10,000). (C) Compilation of trials in which WT fibroblasts were transfected with either vector alone, mutant, or WT Cx43 and GJ assembly monitored without (A) or with (B) 8Br-cAMP.

Figure 6. — GJ assembly following the expression of vector, ^S364PCx43, ^S364ECx43, ^S364ACx43, or ^WTCx43 in WT fibroblasts. (A) Comparison of GJ assembly between WT fibroblasts (10-3) transfected with either ^S364PCx43 or ^S364ECx43 and treated with or without 8Br-cAMP for the entire experiment (*, injected cell). (B) Example of immunoblots of cell homogenates following two different assembly experiments using either WT fibroblasts transiently transfected with ^M257Cx43 or ^S364PCx43. Cells were transfected 48 h prior to experimentation with either vector alone (pIRES; 1, 2, 5, and 6) or mutant construct (3, 4, 7, and 8). Cells were dissociated and recovered for 4 h 15 or 23 min, reaggregated for 15 (^M257Cx43) or 7 (^S364PCx43) min, and treated either without (1 and 3) or with (2 and 4) 8Br-cAMP for the entire GJ assembly experiment. To detect endogenous ^WTCx43 and transfected ^M257Cx43 (arrowhead, first panel) membranes were labeled with NH₂-terminal Cx43 antibody (1:8,000) detected with goat anti–rabbit peroxidase antibody (1:10,000). To detect mutant and ^WTCx43 expression (second panel), membranes were labeled with Cx43 CT antibody (1:8,000; Sigma-Aldrich) detected with goat anti–rabbit peroxidase antibody (1:10,000). (C) Compilation of trials in which WT fibroblasts were transfected with either vector alone, mutant, or WT Cx43 and GJ assembly monitored without (A) or with (B) 8Br-cAMP.