Removal of the SOCS3 SOCS box alters the regulation of the JAK/STAT pathway in vivo. (A) Activation of STAT3 in response to G-CSF. Bone marrow cells from SOCS3^+/+ and SOCS3^ΔSB/ΔSB or SOCS3^ΔVAV/− mice were left untreated (NT) or stimulated with 50 ng/mL G-CSF for 15 minutes, washed, then lysed at intervals between 5 minutes and 120 minutes. Lysates were analyzed by immunoblot using antibodies specific for phospho-STAT3 or total STAT3. (B) Activation of ERK in response to G-CSF. Bone marrow cells from SOCS3^+/+ and SOCS3^ΔSB/ΔSB or SOCS3^ΔVAV/− mice were left unstimulated (0 time point) or stimulated with 50 ng/mL G-CSF for intervals between 5 minutes and 60 minutes. Lysates were analyzed by immunoblot using antibodies specific for phospho-ERK or total ERK. (C) Induction of WT and SOCS box–deleted SOCS3 protein in SOCS3^+/+ and SOCS3^ΔSB/ΔSB bone marrow cells. Cells were left unstimulated (0 time point) or incubated in the presence of 10 ng/mL G-CSF and 10 μM MG132 for 1 to 8 hours and analyzed by immunoblot using antibodies specific for SOCS3 or HSP-70. BM indicates bone marrow.