Figure 5: TDP-43 is required for recruitment of critical spliceosomal components from CBs.

(a) Effect of LPS stimulation on endogenous TDP-43 or coilin interaction with spliceosomal snRNAs. RAW macrophages were stimulated with LPS (80 ng ml⁻¹) for 6 h and then lysed with 2% (v/v) NP-40. Supernatants were subjected to immunoprecipitation with anti-trimethylguanosine (snRNA) antibody before immunoblot analysis with anti-TDP-43 and then with anti-coilin antibody. (b) GFP-TDP-43 co-localizes with endogenous spliceosomal U snRNAs and Sm proteins, but not with SMN. RAW macrophages expressing GFP-TDP-43 were visualized by immunofluorescence microscopy using the indicated antibodies. White arrows indicate colocalization of TDP-43 with snRNAs or Sm proteins. Scale bars, 5 μm. (c,d) LPS stimulation reduced association of TDP-43 with SMN and increased binding of coilin with SMN. Cell extracts from LPS-stimulated RAW macrophages stably expressing HA-TDP-43 or coilin-Myc were immunoprecipitated with anti-HA or anti-Myc antibody, and then subjected to immunoblot analysis with anti-SMN antibody. The same blots were reprobed with anti-HA or anti-Myc antibody to verify the expression levels of TDP-43 or coilin. (e) The association of TDP-43 with coilin was disrupted by LPS stimulation. RAW macrophages stably expressing both HA-TDP-43 and coilin-Myc were incubated with LPS (80 ng ml⁻¹), lysed with 2% NP-40 and then immunoprecipitated with anti-HA antibody before immunoblot analysis with anti-Myc antibody. *immunoglobulin heavy chains, **immunoglobulin light chains. Data are representative of at least three independent experiments.