Fig. 8.

Influence of tau domains and lysine acetylation on SG association of tau. a Schematic diagram illustrating the semi-permeabilized cell assay. After SG induction by MG132, the plasma membrane of Hela P4 cells is selectively permeabilized using digitonin, cytosolic soluble factors are washed out and subsequently nuclear pore complexes blocked by WGA. Next, Alexa 488-labeled tau proteins are incubated with semi-permeabilized cells to allow binding to SGs and after extensive washing, SGs were subjected to immunostaining for the SG makers G3BP1 and TIA-1. b SG association of K18, K25, and hTau40 (400 nM; degree of labeling (“DOL corrected”) = 3.7), scale bar, 10 μm. In the merge, Alexa 488 fluorescence (green) and TIA-1 staining (red) are shown. c Quantification showing the Log2-transformed mean fluorescence intensity of K18, K25, and hTau40 vs. background (bkgr) in SGs. Four replicates with at least 8–10 cells and ≥ 25 SGs each are shown. Error bars correspond to ± SEM. d SG association of unmodified hTau40 (400 nM; DOL = 1.0) in comparison to acetylated hTau40 (Ac-hTau40) monitored by Alexa 488-fluorescence. In the merge, Alexa 488 fluorescence (green) and TIA-1 staining (red) are shown. e Quantification showing the Log2-transformed mean fluorescence intensity of hTau40 and Ac-hTau40 vs. background in SGs. Four replicates with at least 8–10 cells and ≥ 37 SGs each are shown. Error bars correspond to ± SEM. Please note, that due to the wide range of fluorescence intensities of the recombinant proteins in SGs, different laser settings had to be used in b when compared to d in order to avoid pixel saturation and to ensure ideal representation of the differential SG recruitment of the different proteins. Source data are provided as a Source Data file