competes with 20S proteasome for binding with C/EBP leading to its stabilization and Relative mrna levels Supplement Figure S1. Real Time PCR analysis of mrna levels of C/EBPα and PU.1 in wild type (WT) and -null (-/-) mice.
competes with 20S proteasome for binding with C/EBP leading to its stabilization and Relative mrna Levels Supplement Figure S2. Effect of MG132 on mrna levels of C/EBP and PU1. Mouse bone marrow cells were treated with DMSO or 20 M MG132 for 10 hours, RNA was extracted and converted to cdna. Equal amounts of cdna were used with Taqman Master Mix and C/EBPα, PU.1, or GusB Primer and Probe. Final mixture was run on Real Time System (Applied Biosystems) using relative quantitation according to the manufacturer's instructions.
competes with 20S proteasome for binding with C/EBP leading to its stabilization and Overexpression sirna Inhibition Relative mrna Levels * * Relative mrna Levels * * Supplement Figure S3. Effect of on mrna levels of C/EBP and PU1. HL-60 cells were transfected with 0.375 or 0.75 μg -V5 plasmid, and U937 cells were transfected with 100 or 200 nm sirna, 48 hours later, RNA was extracted and converted to cdna. Equal amounts of cdna were run on Real Time System (Applied Biosystems) using relative quantitation according to the manufacturer's instructions. * indicates significant difference.
competes with 20S proteasome for binding with C/EBP leading to its stabilization and input IP: Flag C/EBPa 20s IB: Flag IB: V5 IB: 20S 0.5 1 2 4 -V5 (µl) 0.5 1 2 4 -V5 (µl) Supplement Figure S4. competes with 20S for C/EBPα biding. 1 µg flag C/EBPα and 1 µg -V5 plasmids were used for in vitro translation for 1.5 hours. 5 µl final products of flag- C/EBPα were mixed with 1 µl purified 20S and different amount of -V5 with NADH (5 mm). The mixture were then used for IP using anti-flag, precipitated proteins were probed with antibodies as indicated. All input were 10% of the proteins used in the experiments.
competes with 20S proteasome for binding with C/EBPa leading to its stabilization and 30.0 -Null Gr-1 Positive (Percent WBC) 27.5 25.0 22.5 20.0 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0 3 0 3 -Radiation (Gy) Annexin V- Positive Apoptotic Cells (Percent of Gr-1 Positive Myeloid Cells) 50.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0 3 0 3 -Null -Radiation (Gy) Supplement Figure S5. Bone marrow analysis. Seven to nine weeks old wild type and -null mice were either non-irradiated (0) or irradiated with 3 Grays γ-radiation, waited for 48 hours and analyzed for granulocytes and apoptotic death of granulocytes.
WBC (X1000) competes with 20S proteasome for binding with C/EBPa leading to its stabilization and 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 -Null 0 3 0 3 -Radiation (Gy) Neutrophils (Percent of WBC) 40 -Null 30 25 20 15 10 5 0 3 0 3 -Radiation (Gy) Eosinophils*: Non-irradiated 1.8+0.4 Irradiated 1.9+0.5 -/- Non-irradiated 2.4±0.9 Irradiated 2.2±0.4 Basophils*: Non-irradiated 0.5+0.1 Irradiated 0.4+0.1 -/- Non-irradiated 2.2±0.4 Irradiated 2.1±0.2 Monocytes*: Non-irradiated 1.9+0.4 Irradiated 3.2+0.7 -/- Non-irradiated 2.0±0.8 Irradiated 3.5±0.4 *Percent of WBC Supplement Figure S6. CBC analysis of non-irradiated and 3 Grays γ-radiation irradiated wild type and -null mice. CBC analysis was done 48 hrs post-exposure to radiation. Seven to nine weeks old mice were used.
competes with 20S proteasome for binding with C/EBPa leading to its stabilization and IP IP Input IgG Flag Input IgG V-5 Flag-C/EBP (162-267) -V5 Flag-C/EBP (162-267) 20S IP Input IgG Flag IP Input IgG 20S Supplement Figure S7. Co-IP of Flag-C/EBPα (162-267) with -V5 or 20S. All input were 10% of the proteins used in the experiments.
competes with 20S proteasome for binding with C/EBPa leading to its stabilization and +/+ STRESS -/- STRESS C/EBP 268SGAGAGKAKKSV279 20S C/EBP 268SGAGAGKAKKSV279 C/EBP C/EBP C/EBP C/EBP C/EBP Degradation Myeloid Differentiation & Protection against Myeloproliferative Disease Decreased Myeloid Differentiation & Myeloproliferative Disease 20S Supplement Figure S8. Model to demonstrate protection against stress-induced myeloproliferative diseases.