Question

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 :

Answer 1

You're trying to elicit UBL7 protein expression in HEK293T cells using IFN-beta but see no changes. Consider verifying the activity of IFN-beta, checking cell health, optimizing exposure time and dosage, and confirming the sensitivity of your detection methods. Different cell types and conditions can also affect the response to IFN-beta.

Step-by-step explanation:

You are attempting to study UBL7 protein expression in HEK293T cells and have treated them with various concentrations of recombinant IFN-beta. Unfortunately, you've observed no change in UBL7 levels or the positive control ISG15. There are a few things you might consider in this situation:

• Confirm the activity of your IFN-beta: Make sure that the recombinant IFN-beta is active. It can lose activity if not stored or handled properly.
• Check the health and state of your cells: Ensure that the HEK293T cells are healthy and in the proper state to respond to treatments.
• Consider the timing and dosage: The time of exposure and the amount of IFN-beta used might need optimization. Additionally, ensure that the protein detection methods are sensitive enough to observe the changes.

It's also noteworthy that while interferons, including IFN-beta, are known to alter gene expression to increase resistance to viral infection by reducing cellular protein synthesis, the specifics of the response can vary based on cell type and condition. This might be a factor in why you are not seeing the expected upregulation of UBL7.