NanoBit Luciferase assay

The assay is based on complementation of a split luciferase NanoLuc by the interaction of the tested proteins. For evaluation of the best configuration of the luciferase and TDP-43 fusion proteins, N-terminal and C-terminal fusion have been tested. Plasmid pFN33_TDP-43 and pFN35_TDP-43 yielded the best signal to noise ratio and are both N-terminal fusion of the luciferase large bit (17.6 kDa) and small bit (11 amino acids) respectively. Fusion protein expression is governed by the relatively weak HSV-thymidine kinase promoter, which yields TDP-43 fusion protein levels lower than the endogenous TDP-43 protein in N2a cells (Supplemental Fig. ³), so that cellular processes such as translation and protein sorting may not be overstrained with unphysiological quantities of TDP-43, which could lead to artificial cell behaviour. As a positive control, the interaction of PKA regulatory and catalytical subunit was used, and as a negative control, the absence of interaction with the HaloTag protein was monitored. Quantification of protein self-interaction was measured in the vital N2a cells in 384-well plates by addition of the NanoLuc substrate furimazine (Furimazine stock solution #N1130 1:800 diluted in Optimem, Promega, Madison, WI, USA), 5 µl of which has been added to give a final volume of 50 µl in each well. In our experiments, furimazine provided a stable measurement window of 15–60 min after injection (Supplemental information and Supplemental Fig. ¹). For intermediate washes of the plates and medium change, we used Tecan HydroSpeed^TM (TECAN Group Ltd., Maennedorf, Switzerland). The luminescence signal has been measured in a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany).