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Blood, Vol. 108, Issue 2, 622-629, July 15, 2006
Molecular dissection of Meis1 reveals 2 domains required for leukemia induction and a key role for Hoxa gene activation
Blood Mamo et al.
108: 622
Supplemental materials for: Mamo et al
Immunostaining of intracellular Meis1 and PBX1 proteins. The transduced GP+E cells grown on coverslips (thickness 1D, Fischer scientific) were washed with PBS, and incubated for 10 min. in PBS containing 4% formaldehyde. Following 2 washes with PBS, cells were incubated in PBS with 0.2% Triton-X100 for 5 min, washed 3× with the same solution, and then incubated with primary antibody. All antibodies were diluted to working concentrations in PBS with 0.2% Triton-X100 and 3% bovine serum albumin, fraction V. Following 1 hour incubation with primary antibody, cells were washed 3× in PBS with 0.2% Triton-X100, and incubated for 45 min. with secondary antibody. After 3 washes with PBS with 0.2% Triton-X100, coverslips with cells were overlaid with mounting solution containing 0,25 % 1,4-diazobicyclo[2,2,2]-octane (DABCO, Sigma) and 0.01% diamidino-2-phenylindole (DAPI, Molecular probes, Invitrogen) in glycerol, and placed on a microscope slide.
Primary antibodies used were: rabbit anti Meis1(NT), anti HA (Abcam) and anti VP16 (Beckton Dickinsen), and mouse anti FLAG (M2, Stratagene). Secondary antibodies were Cy3-conjugated anti mouse (Jackson ImmunoResearch) and anti rabbit (Sigma), and Cy5-conjugated anti rabbit antibody (Jackson ImmunoResearch).
Images were acquired at 100-fold magnification using LSM510 Meta confocal microscope and LSM510 Meta V3.2 software.
Files in this Data Supplement:
- Figure S1. Subcellular localization of wild type and mutant Meis1 proteins (JPG, 157 KB)
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Proteins examined are listed to the left of the rows. DAPI denotes nuclear staining, GFP denotes green fluorescent marker protein encoded by retroviral vectors, and DIC denotes differential interference contrast. (1 and 2) In the transduced GP+E cells, the wild type Meis1 and the deletion mutant Meis1Δ334 protein can be found in nucleus and cytoplasm. Meis1 proteins were recognized by rabbit anti-Meis1(NT) antibody and visualized by Cy3-conjugated anti rabbit antibody. (3 and 4) VP16-Meis and VP16-MeisΔ334 preferentially accumulate in the nucleus. VP16 transactivating domain was recognized by rabbit anti-VP16 antibody and visualized by Cy3-conjugated anti rabbit antibody. (5) The FLAG-tagged Meis 1(PIM>MIM) swapping mutant retains a limited nuclear translocation potential. Meis 1FL(PIM>MIM) was recognized by a mouse anti FLAG antibody and visualized by Cy3-conjugated anti mouse antibody. Overlay of DAPI, GFP and Cy3 flourescence is shown in yellow and suggests a correlation between high levels of GFP marker and Meis1FL(PIM>MIM) expression, and between high levels of Meis1FL(PIM>MIM) and nuclear localization of this Meis mutant protein. (6) PBX1HA(MIM>PIM) is predominantly a cytoplasmic protein. PBX1HA(MIM>PIM) was recognized by a rabbit anti-HA antibody and visualized by Cy3-conjugated anti rabbit antibody. Overlay of DAPI and Cy3 fluorecsence shows low levels and speckled distribution of nuclear PBX1HA(MIM>PIM) protein. (7) Co-overexpression of Meis1FL(PIM>MIM) and PBX1HA(MIM>PIM) appears to enhance levels of nuclear PBX1HA(MIM>PIM). Cells presented in (6) were infected with recombinant retroviruses encoding Meis1FL(PIM>MIM) and GFP. PBX1HA(MIM>PIM) was recognized as in (6). See also correlation between high levels of GFP and Meis1FL(PIM>MIM) expression. Overlay of DAPI, GFP and Cy3 flourescence is shown in yellow.
- Figure S2. Southern blot analyses of proviral integrations in DNA isolated from the tissues of leukemic mice (JPG, 54 KB)
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Top panel: clonal analysis of proviral integrations in VP16-Meis and VP16-Meis1Δ334 leukemias. DNA was digested with EcoR I which cuts in each provirus once such that each autoradiographic band represents a uniqe integration event. B, bone marrow; S, spleen; T, thymus. Probe used was a 680 bp fragment encoding GFP cDNA.
Bottom panel: Southern blot analysis of DNA digested with Kpn I, which cuts within LTR and thus releases the integrated provirus.
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