(a) Binding of HTm4 to KAP in yeast two-hybrid assay. Constructs listed in the upper and lower rows were in pGADGH, whereas those to the left of the first column were in pVJLII. HTm4 listed here represents the C_HTm4. MEK, byr, and lamin are negative controls. The positive results are shown as colony growth on selective medium and blue color reaction in the presence of β-gal. (b) Isolation of KAP from U937 and KU812 lysates using GST-C_HTm4 fusion protein. GST-C represents GST-C_HTm4. Lysates derived from U937 and KU812 are absorbed with either GST-C_HTm4 or GST-coupled beads, and bound proteins were analyzed in a Western blot assay for the presence of KAP. (c) Coimmunoprecipitation of HA-tagged KAP and HTm4. CTL, pCMV control vector. The source of the samples is listed on the top. The first two columns on the left are lysates alone and the remaining two are after immunoprecipitation with anti-HA. Samples are analyzed by the Western blot technique using anti-HTm4 for the top two panels and anti-HA for the bottom two. The identified proteins are listed on the right. (d) HTm4 and KAP form a physiological complex. The source of the samples is denoted on the top; the first column from the left is U937 lysate alone; the second and third are after immunoprecipitation with anti-KAP and IgG control, respectively. The presence of HTm4 is indicated on the right after Western blot analysis. Representative figures of at least five experiments.

(a) HTm4 binds to KAP-CDK2 under normal physiological conditions. Samples analyzed are shown on the top. The lane marked anti-CDK2 includes the samples immunoprecipitated with anti-CDK2 mAb and analyzed by Western blot technique using Ab’s as indicated on the left. (b) The C-terminal region of HTm4 is required for its binding to KAP-CDK2. HTm4-Ct, HTm4 without the last 22 amino acids. Ab’s used for the Western blot analysis, after immunoprecipitation with anti-Flag, are listed on the left. The induction of protein expression in the absence of Dox is marked as +, and no induction in the presence of Dox is marked as –. (c) The C-terminal region of HTm4 is required for the enhancement of the phosphatase activity of KAP. The descriptions for c are the same as Figure 4b, except the top panels show the immunoprecipitation with anti-Flag Ab and the bottom panels are derived from 50 μg of total lysate per sample. The upper bands in the lower panels are the dephosphorylated form of CDK2, marked as de-(P), and the lower bands are the phosphorylated (P) form. A reduction in intensity can be seen in the phosphorylated CDK2 in the presence of overexpressed Flag-HTm4. Representative figures of at least five experiments.

Immunoperoxidase staining of secondary follicles of the tonsil with CDK2, HTm4, KAP, and Ki-67. Sections of reactive tonsillar tissue are stained for expression of (a) CDK2, (b) HTm4, (c) KAP, and (d) Ki-67. Note the strong staining of the proliferating cells of the germinal center for all Ab’s with absence of staining of the surrounding mantle zones. The cellular protein localization is predominantly nuclear for CDK2 and Ki-67 (a and d, respectively) with nuclear and paranuclear staining noted for HTm4 and KAP (b and c, respectively). Magnification ×400, HRP stain; hematoxylin was used as counterstain.

The expression of HTm4 is highly regulated during the differentiation of hematopoietic stem cells. (a) MicroRT-PCR analysis of HTm4 expression in single hematopoietic cells at various stages of differentiation. Cell surface markers for each sample are denoted on the top row. G₀ represents the quiescent CD34⁺/CD38^– HSC. The middle row is a Southern blot analysis of HTm4 cDNA obtained through RT-PCR technique. The bottom row shows the loaded cDNA stained with ethidium bromide dye. Each lane represents a single cell. (b) Northern blot analysis of HTm4 expression in CD34⁺ cells that were induced to differentiate with the treatment of either G-CSF or Epo for time intervals as indicated. At day 11, cell populations are determined to be 60% neutrophils and 70% erythrocytes when induced with G-CSF and Epo, respectively.

(a) Colocalization of HTm4 and KAP. Staining was visualized with goat anti-mouse IgG Texas Red–conjugated (left) or goat anti-rabbit IgG FITC–conjugated (middle) secondary Ab’s. Merged image is given on the right showing that HTm4 and KAP colocalized predominately in the perinuclear area. (b) HTm4 is located in the intracellular membrane fraction. Source of the samples are denoted on the top row. Samples shown are in duplicates. The presence of HTm4 is indicated on the right after Western blot analysis. (c) Staining of U937 with anti-HTm4 Ab. The manipulations of samples are denoted on the top. The filled peaks represent the staining with anti-HTm4, and the open peaks represent IgG control. x axis, cell counts; y axis, signal intensity of FITC fluorescence. Representative figures of at least five experiments.

Cell cycle arrest is induced by the exogenous expression of HTm4. The upper panels are U937 with inducible Flag-HTm4 expression vector and the lower panels are with Flag-HTm4-Ct. +, induction of expression in the absence of Dox; –, no induction in the presence of Dox. Time intervals are given on the right. 0 hours, before the addition of FBS into the cultures; 20 hours, cells were cultured in the presence of serum for 20 hours after synchronization. X axis shows cell cycle phase analyzed. Marked here are G₀/G₁ and G₂/M; in between (not marked) is S phase. Representative figures of at least five experiments.