Cx43 is usually present on the plasma membrane of HaCaT cells. Immunofluorescence staining for Cx43 showed the staining in cytoplasm, which is wierd. I tried running western blots with total protein lystates of the HaCaT cells. Most times I did not get any bands at all, rarely I got a faint band. Similarly, I tried overexpressing Cx43 in HaCaT cells. We expected to see gap junction plaques but fluorescence was mostly in the cytoplasmic region.
I dont think there is some thing wrong with my protocol because I tried the same immunofluorescence protocol for Cx43 staining in cervical tissue sections and they were amazing and cx 43 was at the plasma membrane.
What could be the problem with my cells that I am having issues with cx43 ?? Other membrane protein which we were checking expression for, hDlg was perfectly expressed in western blotting, immunofluorescence and overxpression expts.
Hope I am clear...
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