Detection of LDL receptor protein in cultured lymphoblasts by immunoblotting and ligand blotting. (a) Cells from a normolipemic individual (Normal), the index patient in family 1 (FH-1), and the index patient in family 2 (FH-2) were incubated for 16 hours in medium containing 10% (vol/vol) FCS (Serum), 10% (vol/vol) lipoprotein-deficient serum containing compactin (Compactin), or 10% (vol/vol) lipoprotein-deficient serum containing cholesterol and 25-hydroxycholesterol (Sterols). Cell extracts were fractionated on nonreduced SDS-PAGE (50 μg of cell protein/lane) and transferred to nitrocellulose membranes. The membrane was incubated with an anti–LDL receptor mAb (mAb 4B3), followed by a peroxidase-conjugated anti-mouse IgG; bound antibody was detected by chemiluminescence with 60-second exposure to film. The positions of molecular weight markers are shown on the left, and positions of the mature LDL receptor protein and the expected position of the precursor protein (not visible in these cells) are shown on the right. (b) Cells from a normolipemic control, FH-1, a homozygous FH patient with a known mutation in the LDL receptor gene (FH hmz-E387K; ref. 34), and heterozygous FH patients with known mutations in the LDL receptor gene (FH htz-C660S and FH htz-A519T; ref. 3) were preincubated with lipoprotein-deficient serum and compactin and analyzed by ligand blotting with ¹²⁵I-labeled β-VLDL (specific activity = 500 dpm/ng of protein, 1 μg of protein/mL; top) or by immunoblotting with mAb 4B3, as described above (bottom).