Expression of the 20-kDa isoform is common and due to cap-dependent internal translation. (a) Analysis of various cell lines and primary cells confirms the frequent existence of a 20-kDa isoform of Cx43 (GJA1-20k), which includes cervical cancer cells (C33a), mouse embryonic carcinoma cells (NF-1), endometrial cancer cells (Ishikawa), breast cancer cells (BT-549), mouse embryonic fibroblasts (MEFs), lung cancer cells (A549, Hop-62), and primary human fibroblasts (HFs). (b) Knockdown of Cx43 with esiRNA, or by microRNA-1 or −206, shows the concomitant regulation of full-length Cx43 (GJA1-43k) and the GJA1-20k isoform in lung A549 cancer cells. (c) Transfection of Cx43-negative HeLa cells with Cx43 RNA leads to the generation of both full-length GJA1-43k and the GJA1-20k isoform. RNA lacking the 5′ guanosine methylation (CAP), required for standard translation initiation, completely prevents both wild-type (Wt) Cx43 and internal 20-kDa fragment translation. A frameshift (FS) mutation shortly after the Cx43 AUG codon also significantly reduces internal translation, although a 20-kDa band is detected with a very long exposure time (saturating GJA1-43k). (d) Transfection of capped RNA containing a HP7 hairpin loop, preventing cap-dependent ribosomal scanning and translation, completely blocks both GJA1-43k and GJA1-20k expression whereas cap-independent translation of EGFP via the EMCV IRES is maintained (both anti-GFP-HRP and anti-Cx43 antibodies are included in this blot).