Generation of autotaxin-specific monoclonal antibodies and the specific up-regulation of autotaxin protein by EBV-infection of HL cells. (A) Immunoblotting of sf9 cells infected with autotaxin (ATX)-baculovirus using 2A12 antibody demonstrates strong reactivity, whereas sf9 cells infected with wild-type (wt) baculovirus show no reactivity. Also shown here is reactivity against autotaxin in human serum, human plasma, culture supernatant and cells of breast cancer cell line MBA-MD-231, and supernatant and cells of glioma cell line SF539. (B) Western blotting using 2A12 antibody demonstrates increased autotaxin protein in EBV-infected KM-H2 cells (KM-H2 Akata) compared with uninfected KM-H2 cells (left panel). Most other EBV-negative HL cell lines (L428, L540, L1236, HD-MyZ, HDLM2) expressed lower levels of autotaxin protein. (right panel) Down-regulation of autotaxin protein in L591 cells after the loss of the EBV genome. (C) Autotaxin could not be detected in EBV-negative BL cells (Ramos, DG75, BJAB) or in a number of EBV-positive BL cells, including those displaying a type 1 form of EBV latency (Akata, Rael, Elijah), a type 3 form of EBV latency (Raji, Namalwa), or a Wp-restricted pattern of EBV latency (Daudi) that expresses EBNA1 and the EBNA3 family. BL lines are negative for autotaxin, though there is an unidentified lower molecular weight band in the DG75, BJAB, Akata, Elijah, and Rael BL lines and in the IB4 LCL line. Furthermore, of several lymphoblastoid cells lines (X50-7, B95.8 LCL, IB4), only B95.8 cells expressed detectable levels of autotaxin. (D) NPC cells, including EBV-positive C666-1 and EBV-negative HONE-1 cells, lacked autotaxin protein. HONE-1 cells infected with the same Akata-derived recombinant EBV used to generate EBV-positive KM-H2 cells failed to up-regulate autotaxin expression after infection.