Skip to main content
. 2014 Jun;34(12):2264–2282. doi: 10.1128/MCB.01542-13

FIG 3.

FIG 3

COPS5 is stabilized by ATM-mediated phosphorylation. (A) Immunoblot analysis of COPS5 from immunoprecipitates of polyubiquitinated (HA) proteins. Lysates from cells expressing HA-tagged ubiquitin (HA-Ubi) and COPS5 under the unirradiated or gamma-irradiated condition or under the condition of gamma irradiation with ATM kinase inhibitor treatment. Immunoblots of COPS5 and actin are also shown. (B) Immunoblot analysis of MYC-COPS5 from immunoprecipitate of polyubiquitinated (HA) proteins. Lysates from cells expressing HA-tagged ubiquitin (HA-Ubi) and MYC–COPS5-WT, MYC–COPS5- S320A, and MYC–COPS5-D151N without or with gamma irradiation were used. Immunoblots of COPS5 and actin are also shown. (C) Immunoblot analysis for COPS5, PEA15, and p27 proteins in gamma-irradiated HCT116 cells expressing NS or COPS5 shRNAs (shRNA 1 and shRNA 2). Actin was used as a loading control. (D) qRT-PCR analysis of PEA15 mRNA (n = 3) in HCT116 cells carrying NS or COPS5 shRNAs without or with gamma irradiation. Error bars indicate standard errors of the means. (E) Immunoblot analysis of COPS5 and PEA15 levels in HeLa cells or MCF7 cells expressing NS or COPS5 shRNAs without or with gamma irradiation. Actin was used as a loading control. (F) Immunoblot analysis of PEA15 and COPS5 in HCT116, HeLa, and MCF7 cells expressing vector control, COPS5-WT, and COPS5-D151N. Actin was used as a loading control. (G) Immunoblot analysis of PEA15 polyubiquitination (HA) after HA IP from unirradiated or gamma-irradiated cells expressing HA-tagged ubiquitin (HA-Ubi) and GFP-PEA15 in the presence of a nonsilencing (NS) control or COPS5 shRNA. As controls immunoblots for the indicated proteins are shown. (H) Immunoblot analysis of PEA15 polyubiquitination (HA) after GFP-PEA15 immunoprecipitation from cells expressing a vector control or wild-type or mutant (D151N) COPS5 cDNA. The levels of COPS5, GFP-PEA15, and actin are shown as controls.