Nucleoporin 153 Arrests the Nuclear Import of Hepatitis B Virus Capsids in the Nuclear Basket (original) (raw)
Figure 1
Co-immune precipitation of Nup153 from rat nuclear extracts with Immat-C.
A. Importin β binding of Nups after renaturation. Nuclear extract was incubated with importin β and FXFG repeat-containing Nups were precipitated using mAb414-coated biomagnetic beads. Panel A depicts an immune blot using an anti-importin β antibody showing that importin β was co-precipitated by the nucleoporins. 1: mAB414-coated beads + nuclear extract+importin β. 2: mAB414-coated beads without nuclear extract + importin β. 3: Uncoated beads+nuclear extract + importin β. The 54 kDa band present in all lanes represents the heavy chain of the antibodies. The bands at 66 and 47 kDa in lane 1 are most likely degradation products of Imp β. B. Protein co-precipitation by capsids from nuclear extract. Sypro Red stain after SDS PAGE. m: marker. The MW is listed on the left. 1: nuclear extract without precipitation. The migration of different nucleoporins is indicated on the right. 2: Co-immune precipitation of nuclear extract with Immat-C bound to anti capsid antibody-coated biomagnetic beads. 3: Negative control without nuclear extract. 4: Beads only. 5: Co-immune precipitation of nuclear extract without Immat-C by anti capsid antibody-coated biomagnetic beads. The stain shows a precipitation of a protein with an apparent MW of ∼180 kDa. C. Immune blot of the nuclear protein(s), which were co-precipitated by Immat-C. Immune detection was performed using mAb414. 1–5 as in Figure 1B. The blot shows that the co-precipitated protein is mAb414-reactive. D. Phosphoimager scan of 32P-labeled Immat-C (lane 2) and marker (lane 1) showing that a single protein was labeled. E. Phosphoimager scan of precipitations in which 32P-labelled Immat-C was incubated with nuclear extracts followed by precipitation of nucleoporins, which were bound to mAb414-coated biomagnetic beads. Capsid-co-precipitation is indicated by the radioactive core proteins on an SDS PAGE. 1: mAb414-coated beads without nuclear extract + capsids 2: Anti capsid coated beads + nuclear extract + capsids, 3: Anti capsid coated beads + nuclear extract without capsids, 4: Uncoated beads + nuclear extract + capsids. The figure shows that Immat-C precipitates Nup153 from the nuclear extract of rat liver.