Figure 4

RBM45 interacts with TDP-43 and FUS.

(a) FLAG-RBM45 was expressed in HEK293 cells and immunoprecipitations with FLAG antibody or IgG were performed. Live cells were treated with 0.1% formaldehyde to cross-link proteins prior to cell lysis and immunoprecipitation. The crosslinking was reversed by heating in SDS-sample buffer prior to SDS-PAGE. The IP fractions were immunoblotted with TDP-43, FUS, FLAG (FLAG-RBM45) and GAPDH (negative control) antibodies. (b) HA-RBM45 was transfected into HEK293 cells stably expressing FLAG-TDP-43 (left) or FLAG-FUS (right). Immunoprecipitations and immunoblot were performed as described in a. HA-RBM45 was detected with HA antibody. (c) In-cell RNase treatment and crosslinking-IP were performed on cells expressing FLAG-RBM45. The amount of TDP-43, but not FUS, that co-purified with FLAG-RBM45 was reduced with the RNase treatment. (d) In-cell FLAG-RBM45 and TDP-43 interactions as demonstrated by Proximity Ligation Assay (PLA) in the nucleus of HEK293 cells. PLA was performed on HEK293 cells stably expressing FLAG-RBM45. Each red dot indicates the protein-protein interaction (<40 nm) between FLAG-RBM45 and endogenous TDP-43. The primary antibodies are listed on top of each panel. DAPI was used as the nuclear stain. Scale bar: 10 μM. (e) Wild-type and cytoplasmic retained RBM45 have similar binding efficiency with TDP-43. HA-tagged RBM45 wild-type or the NLS M2/3 mutant was transfected into the HEK293 cell line stably expressing FLAG-TDP-43. FLAG-TDP-43 immunoprecipitation and immunoblot were performed as described in b. (f) PLA assays showing the in-cell protein-protein interactions between HA-RBM45 wild-type or NLS M2/3 mutant and endogenous TDP-43 in HEK293 cells. HA-RBM45 constructs were expressed in HEK293 cells and PLA assays were performed. DAPI was used as the nuclear stain. Scale bar: 10 μM. (g) The self-association deficient RBM45 exhibits reduced binding to TDP-43. HA-tagged full-length, D1 and D4 RBM45 constructs were transfected into the HEK293 cell line stably expressing FLAG-TDP-43. FLAG-TDP-43 immunoprecipitation and immunoblot were processed as in b. (h) The self-association deficient RBM45 exhibits reduced binding to FUS. HA-tagged full-length, D1 and D4 RBM45 constructs were transfected into the HEK293 cell line stably expressing FLAG-FUS. FLAG-FUS immunoprecipitation and immunoblot were processed as described in b.