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. 2009 Nov 11;19(2):262–275. doi: 10.1093/hmg/ddp490

Figure 2.

Figure 2.

Consortin interacts with connexins both in vitro and in vivo. (A) Pulldown experiments of different in vitro synthesized and radioactively labeled connexins (Cx26, Cx30, Cx31, Cx32, Cx43 and Cx45) by a batch-purified GST-consortin (short isoform) chimera. For each pulldown assay, three lanes are shown, corresponding to the relevant in vitro translation reaction (input), a control assay with batch-purified GST (GST), and the assay with the appropriate GST chimera (GST-consortin). Bands corresponding to putative connexin monomers and hexamers are indicated by arrows and arrowheads, respectively. Note that the hexamer band is consistently enriched in the GST-consortin pulldown lane, compared with the input lane. (B) In vitro synthesized, radioactively-labeled Cx26 and Cx30 were pulled down by batch-purified GST-consortin-ΔCter synthesized in Escherichia coli, which indicates that connexin binding does not occur through hydrophobic interactions with the putative transmembrane domain of consortin. (C) Pulldown assay of Cx26 from detergent-free lysates of HeLa-Cx26 cells with GST-consortin (short isoform) and GST-consortin-ΔCter chimeras synthesized in E. coli. A band corresponding to putative Cx26 hexamers, detected with an anti-Cx26 antibody, is observed in the GST-consortin-ΔCter lane. In contrast, no band was detected in the GST-consortin lane, indicating that membrane-inserted Cx26 does not interact with the short isoform of consortin. This may be due to a blockade by the transmembrane segment and C-terminal tail of non-membrane-inserted consortin, which are not part of the minimal connexin-interaction region of consortin. (D) Immunoprecipitation of Cx43 from extracts of HeLa cells transiently transfected with expression plasmid pNTCx43. Extracts were immunoprecipitated with an irrelevant monoclonal antibody (anti-myc), anti-Cx43 monoclonal antibody P2D12, or a mixture of anti-Cx43 monoclonal antibodies IF1 and CT1. Bands corresponding to Cx43 and the short isoform of consortin are detected by immunoblotting in the immunoprecipitates obtained with the anti-Cx43 antibodies, but not in those obtained with the irrelevant antibody, indicating that consortin and Cx43 co-precipitate and may interact in vivo. (E) Escherichia coli-synthesized, batch-purified, GST-consortin and GST-consortin-ΔCter do not pulldown in vitro synthesized pannexin 1 (Panx1).