FIGURE 10.

Treatment with lysosome inhibitors has no effect, whereas proteasome inhibitors have a robust effect on Cx46-induced degradation of Cx43. A, RT-PCR analyses of Cx43 mRNA levels in NN1003A cells overexpressing Cx46-EGFP. Total RNA was isolated from stably transfected cells followed by RT-PCR using specific primers for Cx43 cDNA and β-actin cDNA (internal control). The numbers of cycles of PCR are given. Untransfected control and Cx46-EGFP stably transfected cells were treated with lysosome inhibitors chloroquine (200 μm) (B) or leupeptin (200 μm) (C) for 4 h. Cell lysates were prepared, and equal amounts of total protein were analyzed by Western blot using antibody against Cx43CT and β-actin (loading control). D, control or Cx46-EGFP stably transfected NN1003A cells were treated with proteasome inhibitors, ALLN (100 μm) or MG132 (10 μm), for 4 h. Cell lysates were prepared, and equal amounts of total cell protein in each cell lysate were analyzed by Western blot with antibodies against Cx43CT and β-actin (loading control). E, densitometric analyses show that the treatment with ALLN or MG132 restored Cx43 protein levels in Cx46-EGFP-expressing cells. Cx43 and β-actin bands were digitized by UN-SCAN-It gel software. The average pixel value was calculated for Cx43 bands, normalized, and plotted in percent of control (β-actin). *, significant statistical difference (p < 0.05) between indicated data and Cx46-EGFP-expressing untreated cells (**). Data are represented as mean ± S.E. of three independent experiments.