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I'm trying to increase expression of a protein we're attempting to study, UBL7, supposedly unregulated by Type I Interferon and particularly IFN-beta. I've tried treating HEK293T cells (~60% confluence) with 0, 5, 15, 45, 90, 135ng/ml recombinant IFN-beta for 36 hours.

Based on the (quite vague) protocol we're following (Figure 7B) this should be well more than enough to observe a change in both UBL7 and our positive control ISG15. Instead, UBL7 levels are unchanged from 0ng/ml, and no ISG15 is observed. IFN-beta was added directly to DMEM media in a 6-well plate.

I'm quite new to cell culture so if there's some glaring issue please don't hesitate to call me out, I really don't see how no change at all is occurring :( thanks!

Note: the first figure uses PBMCs, but my figure (7b) uses 293T!

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HEK293 cells (whatever the variety) are not an excellent substitute for PBMCs, as they are from a completely different cell lineage and behave very differently in a lot of ways. However, if the paper or protocol you're following uses them, I would also, at first, until you can get your hands on some PBMCs. I don't recall at the moment if 293T cells are sensitive to IFN-$\beta$, but I'll assume for the moment that they are.

I would first test your stock of IFN-$\beta$ to make sure it is active. If ISG-15 is supposed to be upregulated and it isn't, then your IFN treatment isn't working. Get a fresh supply of lyophilized IFN-$\beta$, reconstitute it according to the instructions, aliquot it into single-use portions, store the aliquots at -80, and avoid freeze/thaw cycles. Improper storage can kill cytokine activity. If you must store it at -20, make sure it is a manual defrost freezer (ice forms on the inside over time), as automatic defrost freezers change the temperature and cause the contents to thaw and freeze again. Don't store it at +4 or on wet ice except for during the day while you're using it.

Another line of investigation would be to perform cell counts of the different treatment conditions to ensure that your treatment isn't affecting the cells' growth rate, either positively or negatively. At the same time, determine the protein concentration of your lysates (if you're making them to do your analysis) to ensure they're not different across the conditions.

Finally, while this won't affect your current issue, I would strongly recommend making serial dilutions of your treatment concentrations. 2x, 5x, and 10x are all common dilution schemes. Make your most concentration dilution in one tube, then serially dilute it from that tube into another, then that tube into another, and so on. It helps reduce variability.

Don't worry about things not working the first time. Take a break, think carefully about possible reasons, and correct those issues one at a time so you're not trying to study multiple variables at once. Good luck!

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