Fig. 5. (A) (Upper panel) The reactivity of labeled 11ug/7u RNA in the presence of 18 µg of HeLa nuclear extract (NE) and 18 µg of TDP-43 immunodepleted nuclear extract (NE-TDP-43). (Lower panel) A western blot assay demonstrating that immunodepletion of TDP-43 has occurred. (B) An immunoprecipitation experiment using anti-TDP-43 sera (and its pre-immune sera as control) on labeled 11ug/7u and Δug/Δu RNAs UV cross-linked with 18 µg of nuclear extract. (C) An immunoprecipitation following UV cross-linking of labeled 11ug/7u RNA with increasing quantities of nuclear extract. The arrow indicates the 50–52 kDa immunoprecipitated product whilst the asterisk indicates the second immunoprecipitated band. (D) The effect of UV cross-linking labeled 11ug/7u RNA with GST–TDP-43 alone (50 ng), with nuclear extract alone (18 µg) and nuclear extract mixed with increasing quantities of GST–TDP-43 (10, 25 and 50 ng, respectively). The 50–52 kDa complex and GST–TDP-43 protein are indicated by arrows. (E) A schematic diagram (top) of the recombinant GST–TDP-43(101–261), its expression and purification (left panel), and its reactivity with synthetic 5′ end-labeled (ug)₁₂ RNA following UV cross-linking with (+) and without (–) RNase treatment (right panel). In the third lane (–/+) these two were mixed and loaded together in the same lane. Only 10% of the untreated sample was loaded in the (–) and (–/+) lanes. The lower amount of labeled material in the (+) lane is due to loss of the labeled 5′ end of the synthetic (ug)₁₂ following RNase digestion.