Recently, researchers discovered that treating mammalian cells with juniferdin, a plant-derived compound, affects protein secretion, and have reported that the target of this drug is protein disulfide isomerase (POI). In the following experiment, cultured pancreatic β-cells were treated with juniferdin and protein lysates were isolated and compared to lysates from untreated cells using immunoblot analysis. Probing blots with antibodies against PDI (57 kDa), actin (43 kDa) and pro-insulin (9.8 kDa), show the following:

a. Given that approximately the same amount of protein was loaded in each lane, as evidenced by the actin signals, how do you explain the fact that the PDI levels also appear about the same, while most of the pro-insulin remains accumulated in the juniferdin-treated cells?

b. To confirm your results, protein lysates of juniferdintreated and untreated cells were separated by SDS-PAGE and blotted to membranes and then probed with antibodies against lre1 and Hac1.

Label each blot with the antibody that was used for the analysis. How do you explain the increase in the intensity of the signal seen in the juniferdin-treated cells in blot B?

c. An immunocytochemistry and fluorescence microscopy analysis was undertaken with the antibody used in blot Band a secondary antibody labeled with rhodamine (red). Since juniferdinspecifically affects PDI, a resident rough ER (RER) protein, how do you explain the localization of the signal in the nucleus?

Transcribed Image Text:

Untreated Pro-insulin Untreated PDI 88 60 55 45 23 Juniferdin Juniferdin Untreated Untreated 08 90 75 45 22 Actin Juniferdin Juniferdin