FIG 2.

LamR is required for CSFV infection. (A) Reduced LamR expression following the knockdown of LamR expression by siRNA. PK-15 cells were transfected with siLamR or siScr, treated only with the transfection reagent (Mock), or left untreated (not treated [NT]). The membrane protein (including the 67-kDa LamR) was extracted by use of a ProteoExtract transmembrane protein extraction kit (Merck) and subjected to Western blot analysis. The remaining cell lysate was used for detection of the 37-kDa LamR. Annexin I (a membrane protein) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; a cytoplasmic protein) were included as internal references. (B) Detection of LamR expression on the cell surface of PK-15 cells by flow cytometry. siScr-transfected (siScr) or siLamR-transfected (siLamR) PK-15 cells were incubated with anti-LamR antibodies. PK-15 cells incubated with an irrelevant isotype rabbit antibody (Mock) were used as a negative control. (C) Growth curve of CSFV-N^proFluc in cells transfected with siLamR or siScr molecules. Cells were infected with virus at an MOI of 0.01. (D) Growth curve of the CSFV Shimen strain (CSFV-SM) in cells transfected with siLamR or siScr molecules. The cells were infected with virus at an MOI of 0.01. (E) Growth curve of the CSFV Tianjin/10 strain (CSFV-TJ/10; genogroup 2) in cells transfected with siLamR or siScr molecules. Cells were infected with virus at an MOI of 0.01. (F) Growth curve of the PRV TJ strain in cells transfected with siLamR or siScr molecules. Cells were infected with virus at an MOI of 0.01. (G) Knockdown effects in cells stably expressing shLamR or shScr. (H) PK-15 cell viability tested with an MTT assay. OD₄₅₀, optical density at 450 nm. (I) SK6 cell viability tested with an MTT assay. (J) CSFV infection in PK-15 or SK6 cells stably expressing shLamR (PK-shLamR or SK6-shLamR) or shScr (PK-shScr or SK6-shLamR) or in parent cells. Cells were infected with CSFV at an MOI of 1, and the supernatant was collected at 24 hpi. *, P < 0.05; **, P < 0.01.