1. by Quanzhi Li, Joy L. Frestedt, and John H. Kersey
AF4 Encodes a Ubiquitous Protein That in Both Native and MLL-AF4 Fusion Types Localizes to Subnuclear Compartments
by Quanzhi Li, Joy L. Frestedt, and John H. Kersey
Blood
Volume 92(10):
November 15, 1998
©1998 by American Society of Hematology

2. Schematic representation of predicted AF4 gene structure.
Schematic representation of predicted AF4 gene structure. The relative location and size of C9, C15, and PL12 as compared with AF4 gene are shown as dark lines; alternate splicing sites, breakpoints of t(4;11) translocations, nuclear localization sequences (NSL), and GTP binding motifs of AF4 are indicated. The figure was constructed based on the data from Morrissey et al,2 Frestedt et al,9 Tkachuk et al,11 and Hilden et al.22
Quanzhi Li et al. Blood 1998;92:
©1998 by American Society of Hematology

3. Blocking of anti-C15 (A) and anti-C9 (B) by immnunizing antigens.
Blocking of anti-C15 (A) and anti-C9 (B) by immnunizing antigens. Km3 cell lysate was separated in 8% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The blots were cut into strips and Western blotting was performed using various preparations of primary antibodies. (A) Lane 1, anti-C15 versus cell lysate; lane 2, preimmune IgG; lane 3, anti-C15 blocked by immunizing antigen; lane 4, anti-C15 blocked with GST. (B) Lane 1, anti-C9; lane 2, preimmune IgG; lane 3, anti-C9 blocked by immunizing antigen; lane 4, anti-C9 blocked by GST.
Quanzhi Li et al. Blood 1998;92:
©1998 by American Society of Hematology

4. Immunoprecipitation using anti-C15.
Immunoprecipitation using anti-C15. (A) Immunoprecipitation followed by Western blotting. Lane 1, Km3 cell lysate without immunoprecipitation; lane 2, unlabeled Km3 lysate immunoprecipitated with preimmune IgG; lane 3, Km3 lysate immunoprecipitated with anti-C15; lane 4, anti-C15 with no lysate. The protein samples were separated in 8% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-C15. (B) Immunoprecipitation of radiolabeled proteins. 35S-labeled Km3 lysate (300 μL) was precleared with preimmune IgG and incubated with 1 μg anti-C15 for 3 hours at 4°C. The immune complex was pelleted by protein A/G beads, washed, separated by 10% SDS-PAGE, and autoradiographed.
Quanzhi Li et al. Blood 1998;92:
©1998 by American Society of Hematology

5. Comparison of Western blotting patterns of anti-C15 and anti-C9.
Comparison of Western blotting patterns of anti-C15 and anti-C9. (A) Km3 lysate (20 μg/lane) was separated in 8% SDS-PAGE, transferred to nitrocellulose paper, and probed with anti-C15 (lane 1), anti-C15 plus anti-C9 (lane 2), and anti-C9 (lane 3). (B) K562 lysate (20 μg/lane) was separated in 4% SDS-PAGE, transferred to nitrocellulose paper, and probed with anti-C15 (lane 1) and anti-C9 (lane 2).
Quanzhi Li et al. Blood 1998;92:
©1998 by American Society of Hematology

6. Western blotting patterns of anti-C15 and anti-C9 in 4% SDS-PAGE.
Western blotting patterns of anti-C15 and anti-C9 in 4% SDS-PAGE. Cell lysates were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and probed with affinity-purified anti-C15 (A) or anti-C9 (B). Lane 1, Nalm-6; lane 2, RS4;11; lane 3, Km3; lane 4, Sem-k2; lane 5, K562; lane 6, B1.
Quanzhi Li et al. Blood 1998;92:
©1998 by American Society of Hematology

7. Indirect immunofluorescence microscopy.
Indirect immunofluorescence microscopy. Cultured cells in log phase were harvested and adjusted to 1 to 2 × 105/mL. The cells were attached to microscope slides by cytospin. The cells were probed with anti-C15 followed by goat antirabbit IgG conjugated to FITC. The image of the stained cells was obtained by a Bio-Rad MR C600 confocal microscope and processed using Adobe photoshop software. The cell lines used are indicated.
Quanzhi Li et al. Blood 1998;92:
©1998 by American Society of Hematology