Figure 5

RBM45 homo-oligomerization mediates its incorporation into stress granules and TDP-43 association.

(a) The cytoplasmic granules containing the RBM45 HA-NLS mutant are immunoreactive with stress granule markers TIAR (top) as well as TDP-43 (middle), but are not immunoreactive with FUS (bottom). HA-RBM45 NLS M2 mutant (from Fig. 1) was transfected into SK-N-SH cells and 48 hr post-transfection, the transfected cells were stressed with 1 mM sodium arsenite for 30 minutes followed by immunostaining. HA-RBM45 NLS was immunostained with rabbit anti-HA antibody (green). Nuclei were labeled with DAPI. Scale bar: 10 μm. TIAR was stained with mouse monoclonal anti-TIAR antibody, TDP-43 was stained with mouse monoclonal anti-TDP-43 antibody that recognizes full-length as well as the cleaved C-terminal fragment of TDP-43 and FUS was stained with mouse monoclonal anti-FUS antibody. (b) RBM45-NLS mutant with additional HOA domain deletion (NLS/D5 construct) are not incorporated into cytoplasmic granules and fail to co-localize with TIAR (top) or TDP-43 (bottom). Transfection and immunofluorescence experiments were performed as described in a. (c,d) Quantification demonstrating the percentage of the cells transfected with NLS or NLS/D5 constructs that form cytoplasmic granules positive for TIAR in c or TDP-43 in d (n = 3; cell numbers > 50 per experiment). The p-value was calculated by two-tailed paired t-test. Error bar represents SEM.