Figure 2
Figure 2. Tissue expression, subcellular localization, and dimer formation of Pen. (A) Relative expression of Pen in various murine hematopoietic tissues and lineages. BM and spleen cell subsets were purified by FACS. Quantitative real-time RT-PCR was performed in triplicate and normalized using β-actin as the internal control. The level of Pen expression in FLDS-19 is taken as 1.0. Means ± SD. (B) A multitissue (murine) Northern blot of Pen expression. Each lane contained 1 μg poly(A)-positive RNA. (C) Subcellular localization of pen. BaF3 was transfected with pcDNA3.1/pen-Myc. pen-Myc fusion protein was detected using rhodamine-conjugated anti-Myc MAb (left). (Right) Double staining with DAPI to reveal nuclei. Bar = 20 μM. (D) A schematic representation of pen, pen-Myc, and pen-EGFP fusion proteins. (E) Pen forms disulfide-bonded homodimers. NIH3T3 cells were transfected with expression vectors pcDNA3.1/pen-Myc (labeled as pen-Myc), pEGFP N1/pen (labeled as pen-EGFP), and pcDNA3.1 (negative control) alone or in combination. Cell lysates were prepared 24 hours after transfection and immunoprecipitated with anti-Myc antibodies. The immunoprecipitates were electrophoresed after reduction with β-mercaptoethanol (β-ME) (lanes 1 to 4) or without reduction (lanes 6 and 7), transferred to a PVDF membrane, and probed with anti-Myc antibodies. Under nonreducing conditions, about half of pen-Myc proteins existed as dimers (lane 7, **) and about half as monomers (lane 7, *). Some pen-Myc/pen-EGFP dimers can also be detected (lane 7, *⋄). After reduction, all pen-Myc proteins became monomers (lane 4). The 2 faint bands at about 50 and about 25 kDa positions in lanes 3 and 4 are reduced heavy and light chains of immunoglobulins, respectively. (F) The blot in panel E was stripped and reprobed with anti-EGFP antibodies to reveal pen-EGFP monomers (lanes 2, 4, and 7; ⋄) and pen-Myc/pen-EGFP dimers (lane 7, *⋄). The pen-EGFP monomer in lane 7 represented protein that was noncovalently associated with pen-Myc and therefore coimmunoprecipitated during anti-Myc immunoprecipitation. The lower, 29 kDa band in lane 4 is the unstripped pen-Myc monomer signal.

Tissue expression, subcellular localization, and dimer formation of Pen. (A) Relative expression of Pen in various murine hematopoietic tissues and lineages. BM and spleen cell subsets were purified by FACS. Quantitative real-time RT-PCR was performed in triplicate and normalized using β-actin as the internal control. The level of Pen expression in FLDS-19 is taken as 1.0. Means ± SD. (B) A multitissue (murine) Northern blot of Pen expression. Each lane contained 1 μg poly(A)-positive RNA. (C) Subcellular localization of pen. BaF3 was transfected with pcDNA3.1/pen-Myc. pen-Myc fusion protein was detected using rhodamine-conjugated anti-Myc MAb (left). (Right) Double staining with DAPI to reveal nuclei. Bar = 20 μM. (D) A schematic representation of pen, pen-Myc, and pen-EGFP fusion proteins. (E) Pen forms disulfide-bonded homodimers. NIH3T3 cells were transfected with expression vectors pcDNA3.1/pen-Myc (labeled as pen-Myc), pEGFP N1/pen (labeled as pen-EGFP), and pcDNA3.1 (negative control) alone or in combination. Cell lysates were prepared 24 hours after transfection and immunoprecipitated with anti-Myc antibodies. The immunoprecipitates were electrophoresed after reduction with β-mercaptoethanol (β-ME) (lanes 1 to 4) or without reduction (lanes 6 and 7), transferred to a PVDF membrane, and probed with anti-Myc antibodies. Under nonreducing conditions, about half of pen-Myc proteins existed as dimers (lane 7, **) and about half as monomers (lane 7, *). Some pen-Myc/pen-EGFP dimers can also be detected (lane 7, *⋄). After reduction, all pen-Myc proteins became monomers (lane 4). The 2 faint bands at about 50 and about 25 kDa positions in lanes 3 and 4 are reduced heavy and light chains of immunoglobulins, respectively. (F) The blot in panel E was stripped and reprobed with anti-EGFP antibodies to reveal pen-EGFP monomers (lanes 2, 4, and 7; ⋄) and pen-Myc/pen-EGFP dimers (lane 7, *⋄). The pen-EGFP monomer in lane 7 represented protein that was noncovalently associated with pen-Myc and therefore coimmunoprecipitated during anti-Myc immunoprecipitation. The lower, 29 kDa band in lane 4 is the unstripped pen-Myc monomer signal.

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