Fig. 5. ACF protects apoB RNA from APOBEC1-induced NMD. (A) Northern blot analysis of total RNA from CCL13 cells transfected with 10 µg of plasmid DNA containing β-globin wild-type (wt), PTC 39, 55 or 261 nucleotides of unedited (C) and edited (T) apoB RNA inserted in-frame in exon 2 of β-globin wild type (55C, 55T, 261C and 261T) in the same position as PTC 39. Northern blots probed with β-globin cDNA. Ethidium bromide staining of the 18S RNA from the corresponding gel is shown. (B) Northern blot analysis of total RNA obtained from cells transfected with plasmids 55T or 55C (10 µg) together with 5 µg of lacZ and 0–10 µg of APOBEC1 in a total of 30 µg of DNA per transfection and probed with lacZ, APOBEC1 and β-globin cDNAs. In some experiments, empty vector plasmid DNA was used to make up the total concentration of DNA to 30 µg. (C) Northern blot analysis of total RNA as in (B) except APOBEC1 was replaced with APOBEC1(C96A) catalytically inactive mutant and probed with β-globin cDNA. Other probes are not shown. (D) Northern blot analysis of total RNA from cells transfected with β-globin wild-type (wt), PTC 39, 55T or 55C (10 µg) together with 5 µg of lacZ, 30 µg of ACF and 1–10 µg of APOBEC1 in a total of 55 µg of DNA per transfection, and probed with lacZ, ACF and β-globin cDNAs. Empty vector plasmid DNA was used to make up the total concentration of DNA to 55 µg. (E) Northern blot analysis of total RNA as in (D) except ACF was replaced with ACFΔ55 and probed as in (D). Only the β-globin probe is shown. (F) Northern blot analysis of total RNA from cells transfected with 55T or 55C (10 µg) together with 5 µg of lacZ, 15 µg of APOBEC1 and 10–20 µg of Upf1 wild-type (WT) or R844C dominant-negative mutant (DN) in a total of 50 µg of DNA per transfection and probed with lacZ, Upf1, APOBEC1 and β-globin cDNAs. Empty vector plasmid DNA was used to make up the total concentration of DNA to 50 µg.