Question: You have genetically engineered green fluorescent protein (GFP) containing a KDEL sequence. When the construct is transfected into normal human fibroblasts and examined using fluorescence

You have genetically engineered green fluorescent protein (GFP) containing a KDEL sequence. When the construct is transfected into normal human fibroblasts and examined using fluorescence microscopy, the fluorescence appears throughout the cytoplasm, as drawn on the diagram on the right.

To analyze the results further, fractions of different organelles and the cytoplasm were collected from cells expressing this KDEL-containing GFP construct and then examined on Western blots using antibodies against GFP (27 kDa) and protein disulfide isomerase (PDI), a resident-rough ER (RER) protein of approximately 55 kDa.

The blot confirms the presence of GFP exclusively in the cytoplasm, and as expected a PDI signal in the RER fraction.

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1. How would you explain the PDI band, albeit weak, in the Golgi fraction?

2. Given the function of PDI proteins, what would you expect if both alleles of a PDI gene were knocked out in mice?

Molecular weight ladder Nuclear fraction 90 kDa 72 kDa RER fraction 66 kDa 40 kDa 21 kDa Cytoplasmic fraction Golgi fraction

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