Figure 3

RBM45 forms high-molecular-weight oligomers.

(a) Diagram of the DSS crosslinking experiments. Live HEK293 cells constitutively expressing FLAG-RBM45 underwent in vivo crosslinking using the cell-permeable NHS-ester crosslinker DSS with increasing concentrations from 0 to 5000 μM. The cells were lysed and lysates were run on denaturing SDS-PAGE and probed for immunoblot. (b) Immunoblot analysis of samples generated from a. Samples probed with RBM45 C-terminal antibody showed that FLAG-RBM45 in non-crosslinked cells migrates at the expected molecular weight of a monomer (56 kD), whereas FLAG-RBM45 in DSS-treated cells migrates at the molecular weight of a dimer (~110 kD) or a pentamer (~280 kD). The arrows indicate the sizes of FLAG-RBM45 monomer and oligomers. Samples probed with TDP-43 antibody revealed the TDP-43 monomer (45 kD) and TDP-43 dimer (~90 kD). Samples probed with β-tubulin and GAPDH antibodies were negative control to rule out non-specific crosslinking of cellular monomeric proteins. (c) HA-tagged full-length RBM45 and RRM1-deletion construct (107–474) were expressed in HEK293 cells and DSS in vivo crosslinking (1 mM DSS for 10 minutes) were performed. The oligomerization of the HA-RBM45 constructs was examined by immunoblot and probed with RBM45 C-terminal antibody. Filled arrowheads indicate the monomers, while open arrowheads indicate the pentamers. The observed molecular weights of the monomers and pentamers are indicated next to the bands. The theoretical molecular weights of the monomers and pentamers are indicated below the blot. (d) The HA-tagged D1-D5 constructs (Fig. 2f) were tested for their ability to oligomerize as described in c. Filled arrowheads indicate the monomers, while open arrowheads indicate the pentamers. The theoretical molecular weights of the monomers and pentamers are indicated below the blot. The blot was also probed with TDP-43 (crosslinking positive control) and GAPDH (negative control) antibodies. Full length gels are shown in Supplemental Figure S2b. (e) RBM45 homo-oligomerization occurs in both the nucleus and cytoplasm. The oligomerization of HA-tagged RBM45 or the NLS M2/3 mutant (from Fig. 1) was examined by immunoblot and probed with HA antibody. Arrowheads denote HA-RBM45 and oligomer species. (f) Subcellular fractionation of the HA-RBM45 constructs in e. Fractionation was performed as described in Fig. 1d.