![Figure 5. ASp27 abrogates CD44-induced growth inhibition of NB4 cells. NB4 cells were infected with empty vector- or ASp27 retrovirus-containing medium and treated 2 days later with A3D8 (time 0) as described in “Materials and methods.” (A) Western blot analysis of p27 in empty vector-NB4 cells (controls) and ASp27 cells treated with A3D8 for 0 hours, 24 hours, and 48 hours. Protein levels were evaluated by densitometric scanning, corrected with respect to α-tubulin expression, and expressed relative to the value obtained in empty vector-infected cells at time 0 (arbitrarily 1). These relative protein level values are shown in italics below the lanes. Results represent 1 representative experiment of 3. (B) Empty vector- and ASp27-infected NB4 cells were cultured at 37°C in the absence or presence of A3D8. Cell numbers and viability were evaluated in triplicate by the trypan blue exclusion test. Data are means ± 1 SD calculated using quadruplicate values. Results represent 1 representative experiment of 3. Data obtained in A3D8 and IgG1-treated ASp27-infected cells are nonsignificantly different (P > .05, Mann-Whitney test). (C) Empty vector- and ASp27-infected cells were treated with A3D8 for 48 hours and were tested for BrdU incorporation as described in “Materials and methods.” Values of BrdU incorporation are indicated by the means ± 1 SD calculated using triplicate values. Results represent 1 representative experiment of 3. (D) Empty vector- and ASp27-infected NB4 cells were lysed 0 hours and 48 hours after addition of A3D8. Cyclin E immunoprecipitates were analyzed by Western blotting with specific Abs to cyclin E and p27. Relative protein levels were evaluated as described in panel A and shown in italics below the lanes. Results represent 1 representative experiment of 3. (E) Cyclin E immunoprecipitates were incubated with histone H1 and [γ32P]ATP (5000 Ci [1.85 × 1014 Bq]/mmol; ICN). γ32P incorporation was measured as described in “Materials and methods.” The reaction products were separated by SDS-PAGE, proteins were transferred to nitrocellulose, and phosphorylated proteins were visualized by autoradiography. Phosphorylation was measured in triplicate, relative to controls. Data are the means ± 1 SD from duplicate values. Results represent 1 representative experiment of 2.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/103/3/10.1182_blood-2003-04-1218/6/zh80030455970005.jpeg?Expires=1735551836&Signature=1NKPdOZfNfl0O17mo7OmdUYwOxDrccKjcACvc~syueRl2VDab8UjHmjRuvDhBHfOrvGJCFnDWRlzPbRU1IicTwEvj6JEebg0iJNGhKWlLJJmOyE7-KH0pFI4q8G7URdhihmHmYfACF6T61LmLu6lkYf~aHYC-D~BlKAlGXMcTyyhUF~PJnaDIHNAYwWHnOr0trLRXYbsQZ7BycV605DT0RqYAJsZGzxYTrlDsiYccvkRdTwE7qpP6~OBOnRbZ1L5eI-QYcUzVIUPqz~5MKfXyW71CJhEh-E-7Tytt-Tdkg0Y8lRE6RpFcngabQhgYE9uQx81ARaBlMmO5bMC4xRD7g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ASp27 abrogates CD44-induced growth inhibition of NB4 cells. NB4 cells were infected with empty vector- or ASp27 retrovirus-containing medium and treated 2 days later with A3D8 (time 0) as described in “Materials and methods.” (A) Western blot analysis of p27 in empty vector-NB4 cells (controls) and ASp27 cells treated with A3D8 for 0 hours, 24 hours, and 48 hours. Protein levels were evaluated by densitometric scanning, corrected with respect to α-tubulin expression, and expressed relative to the value obtained in empty vector-infected cells at time 0 (arbitrarily 1). These relative protein level values are shown in italics below the lanes. Results represent 1 representative experiment of 3. (B) Empty vector- and ASp27-infected NB4 cells were cultured at 37°C in the absence or presence of A3D8. Cell numbers and viability were evaluated in triplicate by the trypan blue exclusion test. Data are means ± 1 SD calculated using quadruplicate values. Results represent 1 representative experiment of 3. Data obtained in A3D8 and IgG1-treated ASp27-infected cells are nonsignificantly different (P > .05, Mann-Whitney test). (C) Empty vector- and ASp27-infected cells were treated with A3D8 for 48 hours and were tested for BrdU incorporation as described in “Materials and methods.” Values of BrdU incorporation are indicated by the means ± 1 SD calculated using triplicate values. Results represent 1 representative experiment of 3. (D) Empty vector- and ASp27-infected NB4 cells were lysed 0 hours and 48 hours after addition of A3D8. Cyclin E immunoprecipitates were analyzed by Western blotting with specific Abs to cyclin E and p27. Relative protein levels were evaluated as described in panel A and shown in italics below the lanes. Results represent 1 representative experiment of 3. (E) Cyclin E immunoprecipitates were incubated with histone H1 and [γ32P]ATP (5000 Ci [1.85 × 1014 Bq]/mmol; ICN). γ32P incorporation was measured as described in “Materials and methods.” The reaction products were separated by SDS-PAGE, proteins were transferred to nitrocellulose, and phosphorylated proteins were visualized by autoradiography. Phosphorylation was measured in triplicate, relative to controls. Data are the means ± 1 SD from duplicate values. Results represent 1 representative experiment of 2.
