Characterization of the p14 isoform of NF-E4. (A) Northern analysis of NF-E4. The full-length NF-E4 cDNA was used to probe 20 μg total RNA (lane 1) and 2.6 μg polyA-positive RNA (lane 2) derived from K562 cells. The size standards are indicated. (B) In vitro transcription/translation of p14 NF-E4. The NF-E4 cDNA truncated to codon 101 was cloned into pSP72, and ³⁵S methionine–labeled protein was produced. This protein (lane 2) and K562 cell nuclear extract (lane 1) were resolved by SDS-PAGE on a 12% gel, transferred to PVDF, and immunoblotted with anti–NF-E4 antiserum. The membrane was developed with ECL. (C) Mutation of codon 101 prevents formation of p14 NF-E4. pCAGGS vectors containing FLAG-tagged NF-E4 cDNA truncated to codon 101 and linked to an optimal Kozak sequence (opt-p14-FLAG) (lane 1), or the full-length NF-E4 cDNA with codon 101 unchanged (101ATG-FLAG) (lane 2), or the full-length NF-E4 cDNA with codon 101 mutated to a TTG (101TTG-FLAG) (lane 3) were transfected into 293T cells, and Western analysis of extract from these cells was performed with anti-FLAG antisera. The p22 NF-E4 and p14 NF-E4 isoforms are marked. The band marked with an asterisk was observed with all tagged constructs and presumably reflects a degradation product or an alternatively modified NF-E4 species. The p14 NF-E4-FLAG band (#) was used as the source of protein for sequence analysis. The boxed amino acid sequence was obtained from Edman sequencing of this band. The size markers are indicated. (D) Kozak sequence 5′ to codon 101 of NF-E4. The sequence 5′ to codon 101 of NF-E4 is compared with an optimal Kozak translation initiation site (TIS).⁵³