Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. (a and b) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon membranes, and immunoblotted with anti–c-myc (lanes 1–8) or anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with peroxidase-conjugated anti-mouse IgG and chemiluminescence. (a) Whole-cell extracts (approximately 50 μg of protein per lane) of Chinese hamster ovary (CHO) cells transiently transfected with pcDNA3-c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retroviral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from affected individual 1.1 (ARH1.1 cells) 1 month after infection with c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes from affected individual 1.1 (lane 5). (b) Whole-cell extracts (approximately 50 μg of protein per lane) of EBV-lymphocytes from affected individual 1.1 (lane 6), the same cells 3 months after stable infection with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected cells after preincubation for 16 hours with 0.3 μM trichostatin A (lane 8 and 10). (c) Virus-infected (ARH–/c-myc-ARH) and uninfected (ARH–) EBV-lymphocytes from individual 1.1 were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Data shown are representative of two separate experiments.