Figure 2.
Figure 2. Enforced expression of p14 NF-E4 represses γ- and ϵ-globin gene expression. (A) Diagrammatic representation of the MSCV-based p14 NF-E4 retroviral vector. (B) Western analysis of K562 cell pools transduced with MSCV vectors. K562 cells were transduced with MSCV p14 NF-E4-HA or MSCV alone and GFP-positive cells obtained by FACS. Cells were expanded, resorted, and cultured in 2 pools. Nuclear extract was prepared from MSCV (lanes 1-2) and MSCV p14 NF-E4 (lanes 3-4) and resolved by SDS-PAGE on a 12% gel. The proteins were then transferred to a PVDF membrane that was immunoblotted with anti-HA antisera (top panel) and anti–NF-E4 antisera (bottom panel) and developed by ECL. The p14 NF-E4-HA, p22 and p14 NF-E4 bands, and the molecular weight standards are indicated. (C) Northern analysis of K562 cell pools overexpressing p14 NF-E4. Total RNA was prepared from the pools derived in panel B. Ten micrograms of total RNA from the 2 MSCV pools (lanes 1-2) and 2 MSCV p14 NF-E4-HA pools (lanes 3-4) was analyzed by Northern blot with γ-, ϵ-, and β-globin and NF-E4 probes. GAPDH served as the control. (D) Northern and Western analysis of CD34+ cord blood progenitors transduced with either the MSCV or MSCV-p14 NF-E4-HA retrovirus and expanded in vitro in BFU-E mix for 7 days (see “Materials and methods”). For the Northern blot, 3 μg total RNA was used in each lane, and the probes used are shown on the right. For the Western blot, nuclear extracts were prepared from both samples and resolved by SDS-PAGE using a 12% gel. After transfer to PVDF, the samples were immunoblotted with anti-HA antiserum and developed with ECL. Positions of migration of the p14 NF-E4-HA isoform are indicated. (E) Northern and Western analysis of CD34+ cord blood progenitors expanded in vitro in BFU-E mix for 1, 3, and 7 days (see “Materials and methods”). For the Northern blot, 3 μg total RNA was used in each lane, and the probes used are shown on the right. For the Western blot, cell extracts were prepared from all samples and resolved by SDS-PAGE using a 12% gel. After transfer to PVDF, the samples were immunoblotted with anti–NF-E4 or antitubulin antiserum and developed with ECL. Positions of migration of the NF-E4 isoforms and tubulin are indicated.

Enforced expression of p14 NF-E4 represses γ- and ϵ-globin gene expression. (A) Diagrammatic representation of the MSCV-based p14 NF-E4 retroviral vector. (B) Western analysis of K562 cell pools transduced with MSCV vectors. K562 cells were transduced with MSCV p14 NF-E4-HA or MSCV alone and GFP-positive cells obtained by FACS. Cells were expanded, resorted, and cultured in 2 pools. Nuclear extract was prepared from MSCV (lanes 1-2) and MSCV p14 NF-E4 (lanes 3-4) and resolved by SDS-PAGE on a 12% gel. The proteins were then transferred to a PVDF membrane that was immunoblotted with anti-HA antisera (top panel) and anti–NF-E4 antisera (bottom panel) and developed by ECL. The p14 NF-E4-HA, p22 and p14 NF-E4 bands, and the molecular weight standards are indicated. (C) Northern analysis of K562 cell pools overexpressing p14 NF-E4. Total RNA was prepared from the pools derived in panel B. Ten micrograms of total RNA from the 2 MSCV pools (lanes 1-2) and 2 MSCV p14 NF-E4-HA pools (lanes 3-4) was analyzed by Northern blot with γ-, ϵ-, and β-globin and NF-E4 probes. GAPDH served as the control. (D) Northern and Western analysis of CD34+ cord blood progenitors transduced with either the MSCV or MSCV-p14 NF-E4-HA retrovirus and expanded in vitro in BFU-E mix for 7 days (see “Materials and methods”). For the Northern blot, 3 μg total RNA was used in each lane, and the probes used are shown on the right. For the Western blot, nuclear extracts were prepared from both samples and resolved by SDS-PAGE using a 12% gel. After transfer to PVDF, the samples were immunoblotted with anti-HA antiserum and developed with ECL. Positions of migration of the p14 NF-E4-HA isoform are indicated. (E) Northern and Western analysis of CD34+ cord blood progenitors expanded in vitro in BFU-E mix for 1, 3, and 7 days (see “Materials and methods”). For the Northern blot, 3 μg total RNA was used in each lane, and the probes used are shown on the right. For the Western blot, cell extracts were prepared from all samples and resolved by SDS-PAGE using a 12% gel. After transfer to PVDF, the samples were immunoblotted with anti–NF-E4 or antitubulin antiserum and developed with ECL. Positions of migration of the NF-E4 isoforms and tubulin are indicated.

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