Figure 3.
Southern and Western blot analyses. (A) DNA from LCLs was cut with SacI and MluI, size-separated, and probed by Southern blotting with an EBV MluI DNA (nucleotides 167,129–169,560) which comprises LMP1 DNA. A 2.4-kb band from P3HR-1 (lane 1) and a wt EBV transformed LCL (lane 3) is recognized by this probe. In LCL F-L1 (lane 2), a SacI site near the Flag codons results in a 2.3-kb DNA, whereas the 0.16-kb band ran off the gel. In coinfected LCLs F-ΔL1 to F-ΔL5 (lanes 5–9), Flag LMP1Δ185–211 DNAs have a second SacI site near the last LMP1 codon, resulting in 1.2- and 1.1-kb DNAs, while the 0.16-kb DNA ran off the gel. The 2.4-kb DNA in lanes 5–9 is wt LMP1 DNA. DNA standards are noted on the left. (B) Denatured proteins from 5 × 104 LCL cells were size-separated, blotted to filters, and probed with M5 Flag antibody (Kodak). No signal is detected in P3HR-1 cells (lane 1), in a wt EBV-transformed LCL (lane 3), or in EBV-negative BJAB cells (lane 4). Flag LMP1 (60 kDa) is detected in LCL F-L1, whereas Flag LMP1Δ185–211 (57 kDa) is detected in LCLs F-ΔL1 to F-ΔL5 (lanes 5–9). A protein standard is noted on the left. (C) The same blot was stripped of antibody and reprobed with S12 monoclonal antibody to LMP1. All cells except EBV-negative BJAB (lane 4) demonstrate 60-kDa LMP1 or Flag LMP1.