![Figure 2. Effect of KIT D816V on expression of HO-1 in Ba/F3 cells. (A) Ton.Kit.D816V-27 cells were transfected with a Hsp32 promoter construct (HO-1-luc) and pCMV-βGal as described in “Materials and methods, Hsp32 reporter gene assay.” Cells were starved from IL-3 and kept in control medium or induced to express KIT D816V by exposure to doxycycline (1 μg/mL) for 16 hours. Then, cells were analyzed for luciferase and βGal activities. Luciferase activity was reported as the ratio HO-1-luc/pCMV/βGal and was expressed as a percentage of control. Results represent the mean (± standard deviation [SD]) of 3 independent experiments. (B) Western blot analysis of Ton.Kit.D816V-27 cells after starvation from IL-3 and incubation in control medium or doxycycline (to induce expression of KIT D816V). Western blotting was performed with antibodies against Hsp32 or β-actin (loading control). (C) wt KIT-transfected Ba/F3 cells (Ton.Kit.wt) were starved from IL-3 and kept in control medium or were induced to express KIT D816V by exposure to doxycycline. To activate wt KIT, cells were kept in SCF (100 ng/mL). After incubation, cells were analyzed for Hsp32 reporter gene activity. Results represent the mean (± SD) of 3 independent experiments. (D) Western blot analysis of Ton.Kit.wt cells after starvation from IL-3 and incubation in control medium, doxycycline (to induce expression of KIT), or doxycycline and SCF (100 ng/mL). (E) Western blot analysis of expression of Hsp32 in cultured (21 days) human blood cell-derived mast cells (d21 MC), KU812 cells (KIT+), MO7e cells (KIT+), U937 cells (KIT−), and K562 cells (KIT−), after exposure to control medium, SCF (100 ng/mL), or hemin (10 μM) at 37°C for 24 hours. Western blotting was performed with antibodies against Hsp32 or β-actin (loading control). (F) KU812 cells were exposed to control medium (control) or SCF (100 ng/mL) for 24 hours. Then, lysates were prepared and subjected to immunoprecipitation with anti-KIT antibody 1C1 followed by Western blot analysis using 4G10 antibody (p-KIT) or 1C1 antibody (controlling total KIT expression). In the same experiment, cell lysates were also subjected to Western blot analysis using antibodies against Hsp32 and β-actin.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/110/2/10.1182_blood-2006-10-054411/4/zh80140704870002.jpeg?Expires=1722835511&Signature=FTJ2WYfa7MtSW0D8FIm5Qe-yCmkCnnVF7gam3UoPTSagjtg1OoySladB~LoOXv3D0c7Zd~vQ-Gl-LsyRJ9OSkvdu7k5MCWRfTUkF4KklonuAv7nl-gmWKIkTbDGxoRxpB-bCDwy-IrjzueJiP30SI91BDoV2TSEmNMl2xqenwLw~DJpadSZqeAs8oS9vBnJF9Lx12UBzXOMCZUuwNFoPUAdZTmUxqP5xWKsApCdRZFbJfIPWpFsgbixwWxoPjhBqJEfsdN6KcAQhFC1r1maCJRN~TSxRe6YnQjFce4OmOc9lpO8hB78IcRLeaIpuHbT2I0-8HOuczxYpl~eZrwk9eA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of KIT D816V on expression of HO-1 in Ba/F3 cells. (A) Ton.Kit.D816V-27 cells were transfected with a Hsp32 promoter construct (HO-1-luc) and pCMV-βGal as described in “Materials and methods, Hsp32 reporter gene assay.” Cells were starved from IL-3 and kept in control medium or induced to express KIT D816V by exposure to doxycycline (1 μg/mL) for 16 hours. Then, cells were analyzed for luciferase and βGal activities. Luciferase activity was reported as the ratio HO-1-luc/pCMV/βGal and was expressed as a percentage of control. Results represent the mean (± standard deviation [SD]) of 3 independent experiments. (B) Western blot analysis of Ton.Kit.D816V-27 cells after starvation from IL-3 and incubation in control medium or doxycycline (to induce expression of KIT D816V). Western blotting was performed with antibodies against Hsp32 or β-actin (loading control). (C) wt KIT-transfected Ba/F3 cells (Ton.Kit.wt) were starved from IL-3 and kept in control medium or were induced to express KIT D816V by exposure to doxycycline. To activate wt KIT, cells were kept in SCF (100 ng/mL). After incubation, cells were analyzed for Hsp32 reporter gene activity. Results represent the mean (± SD) of 3 independent experiments. (D) Western blot analysis of Ton.Kit.wt cells after starvation from IL-3 and incubation in control medium, doxycycline (to induce expression of KIT), or doxycycline and SCF (100 ng/mL). (E) Western blot analysis of expression of Hsp32 in cultured (21 days) human blood cell-derived mast cells (d21 MC), KU812 cells (KIT+), MO7e cells (KIT+), U937 cells (KIT−), and K562 cells (KIT−), after exposure to control medium, SCF (100 ng/mL), or hemin (10 μM) at 37°C for 24 hours. Western blotting was performed with antibodies against Hsp32 or β-actin (loading control). (F) KU812 cells were exposed to control medium (control) or SCF (100 ng/mL) for 24 hours. Then, lysates were prepared and subjected to immunoprecipitation with anti-KIT antibody 1C1 followed by Western blot analysis using 4G10 antibody (p-KIT) or 1C1 antibody (controlling total KIT expression). In the same experiment, cell lysates were also subjected to Western blot analysis using antibodies against Hsp32 and β-actin.
