![Fig. 2. Reduction of phosphorylation of endogenous STAT5 by ▵STAT5 expression. / Lysates from Ba/F3p210ΔSTAT5 cells (maintained in either the presence or absence doxycycline [1 μg/mL] for 24 hours) were incubated with anti-STAT5 antibody C17 and protein G beads for 3 hours and then subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Probing with an anti-STAT5 antibody raised against the SH2 and SH3 domains of STAT5 revealed equal amounts of STAT5A and STAT5B (middle panel). When probed with antiphosphotyrosine antibody 4G10, STAT5 was found to be constitutively phosphorylated in both Ba/F3p210Ctrl cells and Ba/F3p210ΔSTAT5 cells (upper panel). Induction of ΔSTAT5 for 24 hours was associated with a reduction of tyrosine phosphorylation of STAT5. A longer exposure of the blot revealed that ΔSTAT5 coprecipitated with endogenous STAT5 in Ba/F3p210ΔSTAT5 cells (lower panel). Neither endogenous STAT5 nor ΔSTAT5 coprecipitated when lysates were incubated with normal rabbit serum and beads.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/95/6/10.1182_blood.v95.6.2118/6/bloo00605002w.jpeg?Expires=1735558496&Signature=fGiW2-y~3HP4j4ghB9vcGoAnJG63bkTlW4r-Z7t8MlkfwG3A-SVL-M69aXNQsGdD6jvAJLhim8xPc62vHKjjtwh0iFOxbWml9scAM4n9VwSjUPpQ6iyGRwmcUffg2NdAsG2OVMXdUBjodng1AqHIVGO-UsTOVwM~7g5jCGeN4JbNPyc516ZC8ZdtmGuqGlzzglj3Y~15-8GE3KdToomrwhLf2ReyhAYgFBo7TVIpqFknEfUNU9enOZTHCgH6tCH~byjM1WlvRX-I71dc2KGXJ85P09mzWN5ABXl2nUxX85U~yKHhCyQg5MlIZ0QwJoEzMpevbBVpc6CRUguzyfF9Dw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Reduction of phosphorylation of endogenous STAT5 by ▵STAT5 expression.
Lysates from Ba/F3p210ΔSTAT5 cells (maintained in either the presence or absence doxycycline [1 μg/mL] for 24 hours) were incubated with anti-STAT5 antibody C17 and protein G beads for 3 hours and then subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Probing with an anti-STAT5 antibody raised against the SH2 and SH3 domains of STAT5 revealed equal amounts of STAT5A and STAT5B (middle panel). When probed with antiphosphotyrosine antibody 4G10, STAT5 was found to be constitutively phosphorylated in both Ba/F3p210Ctrl cells and Ba/F3p210ΔSTAT5 cells (upper panel). Induction of ΔSTAT5 for 24 hours was associated with a reduction of tyrosine phosphorylation of STAT5. A longer exposure of the blot revealed that ΔSTAT5 coprecipitated with endogenous STAT5 in Ba/F3p210ΔSTAT5 cells (lower panel). Neither endogenous STAT5 nor ΔSTAT5 coprecipitated when lysates were incubated with normal rabbit serum and beads.
