The specificity of the Cxs 26, 32 and 43 antibodies used here was shown by their binding to the parent connexins in Western blots prepared from brain, liver and heart samples; they detected bands corresponding to 22, 27 and 43 kDa proteins respectively (Fig. 4.1-4.3).
Analysis of samples of visual cortex of PO, P7, P14 and P21 rats showed the binding of the Cx 26 antibody to its parent protein (Fig. 4.1a). Similarly, Cxs 32 and 43 antibodies also detected their corresponding parent proteins (Fig. 4.1b,c) The
pattern of relative expression of connexins in Western blots mirrored closely the developmental profile of these antigens observed in immunolabelled sections from brains of corresponding ages. Connexin 26, present at low levels at PO, showed a dramatic increase in the next 2 weeks, an observation consistent with the immunocytochemical picture. Bands of 27 and 43 kDa, corresponding to Cxs 32 and 43, were detected at low levels at P7 and thereafter showed higher levels of expression with development. In addition bands were not detected in blots incubated with preadsorbed antibodies.
Immunoblots of membrane-enriched fractions obtained from the adult cerebellum and frontal and visual cortices showed bands corresponding to Cxs 32 and 43 (Figs. 4.2, 4.3). Densitometric analysis of these bands showed that the visual cortex had twice the amount of Cx 32 present in the frontal cortex, but the latter had 50% more Cx 43. These results confirmed the regional differences in the distribution of connexin immunoreactivities observed in tissue sections.
4.4.2. Northern Blotting
Analysis of total RNA extracted from visual cortices at PO, 14, 28 and 42 rats by Northern blotting showed bands corresponding to transcripts of Cxs 26 (2.8 kb), 32 (1.6 kb) and 43 (3 kb) (Fig. 4.4). Densitometric analysis (Fig. 4.5.) of these bands was in close agreement with the immunocytochemical and immunoblot results of the differential expression of the three connexins during postnatal development. Thus, Cx 26 mRNA was abundant between birth and P14, whereas Cxs 32 and 43 mRNA were present from as early as PO and increased with age.
Fig. 4.1. Localization of connexins in Western blots of control (liver or heart) and cortical tissues: Western blots of samples labelled for Cxs 26 (a), 32 (b) and 43 (c); lane 1 corresponds to samples of control tissues (liver, Cxs 26 and 32; heart, Cx 43), and lanes 2-5 correspond to samples of visual cortex of rats at PO, P7, P14 and P21. (a) Connexin 26 antibody detected bands corresponding to Mr 22 kDa protein in liver and visual cortex of PO, P7, P14 and P21 rats, with occasional faint labelling of bands at higher molecular weights. Note the increase in the intensity of signal between PO- P14 (lanes 2-4). (b) Connexin 32 antibody detected bands corresponding to Mr 27 kDa in liver and visual cortex of P7, P14 and P21 animals. Note the absence of signal at PO (lane 2). (c) Connexin 43 antibody detected a prominent band at Mr 43 kDa in heart and brain samples, in addition to a band at Mr 66 kDa as reported by Beyer et al. (1989). The position of the molecular mass markers is given on the left.
f; i - 200- 97- 66— 46“ 30- 2 2- 14— 200- 97“ 66— 46— 30“ _ 2 2- 14- 200. 97- 66- 46" 3 4 5 30- 22- 14
Fig. 4.2. Localization of Cx 32 in Western blots of samples of adult liver, cerebellum, visual and frontal cortices, (a) Connexin 32 antibody detected bands corresponding to 27 kDa protein in liver and brain samples, with occasional faint labelling of bands at higher molecular weight; (b) the same samples treated with preadsorbed antibody (40 /xg/ml of relevant peptide) showed no labelling at the expected positions. Note the relative abundance of connexins in visual and frontal cortices. Connexin 32 labelling was more abundant in visual cortex (a-lane 3). The position of molecular mass markers is given on the left. Lanes 1, 2, 3, 4 correspond to samples of cerebellum, frontal cortex, visual cortex and controls (liver)
respectively.
20 0- 9 7 .4 - 66.0— 4 6 .0 — 30.0 — 2 1 .5 - 14.3—
Fig. 4.3. Localization of Cx 43 in Western blots of samples of adult heart, cerebellum, visual and frontal cortices, (a) Connexin 43 antibody detected a
prominent band at 43 kDa in heart and brain samples, in addition to a band at 66 kDa as reported by Beyer et al., 1989 and Green and Severs, 1993; (b) treatment of the same samples with preadsorbed Cx 43 antibody (40 jxg peptide/ml) blocked the labelling of bands at the expected positions. Note the relative abundance of connexins in visual and frontal cortices. Connexin 43 showed higher abundance in frontal cortex (a-lane 2). The position of molecular mass markers is given on the left. Lanes 1, 2, 3, 4 correspond to samples of cerebellum, frontal cortex, visual cortex and controls (heart) respectively.
2 0 0- 9 7 . 4 - 66.0 — 46.0 —
4
1
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4
3 0 . 0 - 2 1 . 5 - 14.3 —Fig. 4.4. Northern blots of samples labelled for Cxs 26, 32, 43 and GAPDH transcripts; lane 1 corresponds to control tissues (liver or heart), and lanes 2-5 correspond to samples of visual cortex of rats at PO, P14, P28 and P42. (A) Connexin 26 antisense RNA probes detected a band corresponding to 2.8 kb in liver and brain samples. Note the abundance of Cx 26 mRNA between P0-P14. (B) Connexin 32 riboprobes detected a band corresponding to 1.6 kb in liver and brain samples. (C) Connexin 43 antisense probes detected a 3.0 kb band in heart and brain samples, (d) GAPDH antisense RNA probes were used as an internal control.
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3
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Fig. 4.5. Histogram illustrating the differential expression and relative abundance of connexins mRNA in the visual cortex at four stages of postnatal development
Relative abundance of mRNA