Figure 2.

Characterization of LMP1 in infected LCLs. (A) Southern blot analysis of EBV recombinant infected LCLs. DNA cut with SacI and MluI was Southern blot-probed with EBV MluI DNA (nucleotide 167,129 to 169,560), which comprises LMP1 DNA. A 2.4-kb DNA from P3HR-1 cells and a wild-type EBV-transformed LCL is hybridized by the probe. In LCLs F-LMP1 1.1, 1.2, and 2.1, which are infected with an F-LMP1 recombinant from F-LMP1–1 or F-LMP1–2 LCL, a SacI site near the Flag codons results in a 2.3-kb DNA whereas the 0.16-kb DNA ran off. In coinfected LCLs F-LMP1-ID 1–4 or in singly infected LCLs F-LMP1-FFD 1.1–1.6, which are infected with an F-LMP1-FFD recombinant from F-LMP1-FFD-1 LCL, F-LMP1-ID, and F-LMP1-FFD DNAs, have a second SacI site near the last codon, resulting in 1.2- and 1.1-kb DNAs whereas the 0.16-kb DNA ran off. The 2.4-kb DNA in LCLs F-LMP1-ID 1–4 is P3HR-1 LMP1 DNA. Markers in kb are at left. (B) Immunoblot analysis of LMP1. Proteins from 5 × 10⁴ cells were size-separated, blotted to filters, and probed with antibody to Flag. A 60-kDa band in LCLs F-LMP1 1.1, 1.2 and 2.1, F-LMP1-ID 1–4, and F-LMP1-FFD 1.1 and 1.2 is Flag-LMP1 (wild type or mutant). A standard in kDa is noted at left. (C) Immunofluorescent staining of cells with antibody to Flag. (C1) F-LMP1-ID-1, an LCL coinfected with P3HR-1 and F-LMP1-ID recombinant. (C2) F-LMP1 1.1, an LCL infected with F-LMP1 recombinant only. (C3) P3HR-1 that expresses LMP1. (C4) LCL infected with wild-type EBV that expresses LMP1 without Flag.