FGF2 and FGF4 export assay
This protocol is extracted from research article:
Escherichia coli as a platform for the study of phosphoinositide biology
Sci Adv, Mar 27, 2019; DOI: 10.1126/sciadv.aat4872

To assay export of FGF2, we expressed a small luciferase, nanoLuc, at its C terminus. The nanoLuc substrate is not membrane permeable; thus, signal should be greatly increased upon export of the FGF2-nanoLuc fusion protein to the outside of the plasma membrane. We performed these experiments on several mutants that should diminish or enhance the export of FGF2 and of FGF4 (see Results).

For these experiments, cells were grown in tubes containing variable amounts of inositol for 3 hours, after which 50 μl of culture was pipetted to a well of a 96-well plate. This was diluted with 30 μl of freshly made phosphate-buffered saline (PBS; pH 7.0) containing 30 μl of substrate for nanoLuc (N113A, Promega) per 10 ml of buffer. At this point, OD600 and luminescence were measured on a plate reader giving the background level of luminescence for each treatment. Notably, because of the dilution and the different path length, this would not be the OD600 as it would be standardly measured in the culture, but simply a linear function of OD600, useful to identify gross errors in pipetting and to verify lysis after the addition of lysis buffer. After the measure of the background luminescence, or prelysis luminescence, we added 100 μl of PBS (pH 7.0) with BugBuster such that the final concentration of BugBuster in the mix would be 1×. Lysis was allowed to run for 15 min and confirmed by the decrease of the OD600 reading in the well. After lysis was confirmed, 20 μl of freshly made PBS (pH 7.0) containing 20 μl of substrate per 10 ml of buffer was added to each well and luminescence was measured again, resulting in the postlysis measurement. All measures were performed immediately after substrate addition to guarantee substrate excess and the validity of the measurement. The pre- and postlysis measures are not directly equivalent since the enzyme is in a different environment for each case. Thus, the usefulness of the measurement is not in its absolute value but in the pattern of the values observed for the different constructs.

For the analysis, we used an index created by dividing the value of prelysis luminescence times 100 over the postlysis luminescence. This can be viewed as a percentage of total luminescence present before lysis. Note that if the activity of the nanoLuc is different in the cytosol compared to the media used for lysis, this is may not reflect an absolute fraction of luminescence in the cytosol. However, since we used the same external buffer in all experiments, it does reflect the relative levels of cytosolic luminescence between different experiments. The values obtained in this way were then used to calculate a three-way ANOVA and a post hoc Tukey HSD test.

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