FIGURE 5.

In vivo expression of HMBs is responsive to ischemia/hypoxia. Juvenile rats respond with a significant increase of the HMB₀ form after ischemia. A, HMBs of brains from untreated animals (lane I) show faint reactions at the height of HMB₀ and a doublet band at HMB₂, whereas the treated site reveals a robust reaction with dominance of the HMB₀ form (lane II). B, mean values of the accumulated protein of all three bands indicate a significant increase (**, p < 0.01) in total protein level after ischemic/hypoxic treatment. n = 4 independent experiments. C, phosphatase treatment confirms that HMB₁ and HMB₂ are phosphoforms of HMB₀. Lane I, protein extract from NIH3T3 cells treated with PIPES buffer, omitting phosphatase enzymes and incubated at 37 °C for 60 min (Ctrl). Lane II, NIH3T3 protein extract treated with potato acid phosphatase and incubated at 37 °C for 60 min. Phosphatase treatment resulted in the loss of P1 and P2 phosphoforms of full-length Cx43 as compared with phosphatase-omitted extract (lane I). Moreover, phosphatase treatment reveals loss of HMB₁ and HMB₂ and persistence of HMB₀. D, Western blot of amino-terminally tagged HA-Cx43 shows no lower fragments of HA-Cx43. Lane I, untransfected NIH3T3 protein extract. Lane II, extract from HA-Cx43-transfected NIH3T3 shows full-length Cx43 isoforms (P0, P1, and P2). Further HA-Cx43 bands in the lower range are not detected by anti-HA antibodies, indicating that protein truncation of full-length Cx43 is unlikely to be responsible for the generation of high mobility bands. Error bars, S.E.