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. 2013 Dec;87(23):12675–12684. doi: 10.1128/JVI.01497-13

Fig 6.

Fig 6

Zn2+ ion-dependent JAB1-Δ2 LHBS association in human cell lysates. (A) The wild-type (W) and Δ2 LHBS stably expressed in HuH7 cells were immunoprecipitated in the presence of 20 μM Zn2+ ions with an antibody that recognizes hemagglutinin (HA) tagged to LHBS. The immunoprecipitants were probed, using Western blotting (WB), with antibodies that recognize HA and JAB1. V, cells containing the gene expression vector pQCXIP. (B) Expression of HBs and HBc RNA and proteins in HuH7 cells transfected with pHBV1.3 (W), pHBV1.3/Δ2LHBS (Δ2), or the vector control (V) by RT-PCR, Northern blotting (with digoxigenin-labeled HBx gene as a probe), and Western blotting. In Western blotting, the HBS proteins were detected with two different antibodies: the total HBS and LHBS alone were detected using the antibodies recognizing the major S and the pre-S1 regions, respectively. (C) Coimmunoprecipitation assays were performed to detect the association of JAB1 with the wild-type and Δ2 LHBS in HuH7 cells transfected with pHBV1.3 (W), pHBV1.3/Δ2LHBS (Δ2), or the vector control (V) in the presence of 5, 10, and 20 μM Zn2+ ions and EDTA (1 mM). The mouse monoclonal antibody recognizing the pre-S1 region of LHBS was used to immunoprecipitate the LHBS-associated protein complex, which was then analyzed by Western blotting (WB). The levels of JAB1 immunoprecipitated with LHBS from at least three experiments were quantified, as shown as a bar chart below the WB images. (D) Coimmunofluorescence assays were performed to detect the localizations of JAB1 with the wild-type (WT) and Δ2 LHBS (Δ2) or its H71Y/H116Y double mutant (Δ2 H71Y/H116Y). The bar chart below the cell images indicates the relative JAB1 levels colocalized with LHBS, detected in at least three independent experiments. *, P < 0.05. (E) Coimmunoprecipitation assays were performed to detect the association of JAB1 with Δ2 LHBS and its H71Y and H116Y mutants and the H71Y/H116Y double mutant, using the same method as described for panel A. (F) Levels of p27Kip1 affected by the wild-type and Δ2 LHBS and its respective mutants, detected using WB. HA and JAB1, WB shows the levels of LHBS-HA and endogenous JAB1. Tubulin was the loading control.