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. 2013 Apr 15;110(18):7306–7311. doi: 10.1073/pnas.1214441110

Fig. 1.

Fig. 1.

Soluble LDLR binds VSV and inhibits infection by VSV and transduction by a VSV-G-pseudotyped lentiviral vector. (A) Survival ± SD of WISH cells as determined by Neutral red staining after treatment with sLDLR and challenge by VSV at the indicated MOI. n = 3. (Inset) SDS/PAGE of sLDLR (10 µg). Molecular mass markers (kDa) are shown on the right lane. (B) Surviving WISH cells, bovine MDBK cells, and murine L cells after treatment with serially twofold-diluted sLDLR (starting at 8 µg/mL) followed by VSV (MOI = 1 for WISH and MDBK cells, MOI = 0.07 for L cells. C, no virus; V, VSV without sLDLR. (C) Surviving WISH cells after addition of sLDLR (1 µg/mL) at the indicated times relative to the time of VSV (MOI = 0.1) addition. In well R, sLDLR was added for 120 min and removed before VSV challenge. C and V are as in B. (D) Quantitative RT-PCR of VSV RNA after attachment of VSV (MOI = 10) at 4 °C for 4 h to WISH cells in the presence of the indicated sLDLR concentrations. VSV RNA ± SE is normalized to TATA binding protein mRNA; *P < 0.02, **P < 0.002, compared with the leftmost bar, n = 3. (Inset) RT-PCR products of VSV RNA, isolated after similar experiments, performed at 4 °C and at 37 °C. (E) Surface plasmon resonance analysis of VSV binding to immobilized sLDLR in PBS with or without CaCl2 (1 mM). (F) Surface plasmon resonance analysis of sLDLR binding to immobilized VSV-G-LV in PBS + 1 mM CaCl2. (G) (Upper) Immunoblotting of VSV-G after coimmunoprecipitation of a solubilized VSV-sLDLR complex with the following antibodies (lanes): 1, mAb 28.28 anti-LDLR; 2, mAb C7 anti-LDLR; 3, isotype control mAb; 4, no antibody. A VSV-G marker is shown in lane 5. (Lower) Reblotting of the membrane with anti-LDLR mAb 29.8. (H) EGFP expression (green) after transduction of FS-11 fibroblasts with either EGFP-encoding VSV-G-LV or EGFP-encoding LCMV-LV in the presence or absence of sLDLR (5 µg/mL). Nuclei were counterstained with Hoechst 33258 (blue). (Insets) Enlarged magnifications. (I) Average ± SD EGFP expression in cultures transfected as shown in H. ***P < 0.003, n = 4. N.S., not significant (P = 0.525), n = 4.