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. 2019 Aug 14;9(21):6334–6353. doi: 10.7150/thno.36818

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This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

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The ATF4-Cbl-c-RET complex leads to RET degradation. A, ATF4 promotes in vivo ubiquitination of RET but not ΔTK RET. Expression vectors encoding HA-ATF4, Myc-RET mutants and FLAG-ubiquitin were transfected into U-2 OS cells as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody, and polyubiquitinated RET was detected by immunoblotting with anti-FLAG antibodies (upper panel). Expression level of each protein was assessed by anti-Myc, anti-HA, and anti-GAPDH antibodies (lower panel). B, Detection of the interaction between ATF4 and RET in vitro. Glutathione-agarose beads containing GST or GST-ATF4 were incubated with whole-cell extracts derived from U-2 OS cells expressing WT RET or ΔTK, C634W, K758M and M918T mutants. C, U-2 OS cells were transiently transfected with expression vectors encoding WT ATF4, WT RET, and ΔTK RET. After 24 h, the cell lysates were analysed by western blotting using antibodies against the indicated epitope tags. D and E, Coimmunoprecipitation of FLAG-ATF4 and HA-Cbl-c in U-2 OS/BTZ cells treated with 20 μM MG132 for 4 h. Overexpression of ATF4 increased the binding between RET and HA-Cbl-c (D), whereas knockdown of ATF4 decreased their binding (E). F and G, U-2 OS cells were cotransfected with HA-Cbl-c, Myc-RET, and FLAG-ubiquitin, and His-ATF4 (F) or siATF4 (G), and protein extracts were immunoprecipitated. Ubiquitination of RET was measured with an anti-Myc antibody. Cell lysates were immunoblotted with the indicated antibodies. H, U-2 OS and U-2 OS/BTZ cells transfected with increasing concentrations of the FLAG-ATF4 expression vector for 24 h were immunoblotted for endogenous proteins with the indicated antibodies. I, Expression of HSPA5 and CBLC in control vector- and FLAG-ATF4-transfected OS and OS/BTZ cells analysed by qRT-PCR. J, ATF4 binds to the promoter. U-2 OS and U-2 OS/BTZ cells were transfected with ATF4 or control vector. Chromatin immunoprecipitation was performed using either a control IgG antibody or an antibody against ATF4. PCR primers were designed to amplify the specific CBLC promoter fragment spanning from -382 to +1. Primers for the DDIT3 promoter were used as a positive control. Bars represent the mean ± SD, *P < 0.05.

Inline graphic — The ATF4-Cbl-c-RET complex leads to RET degradation. A, ATF4 promotes in vivo ubiquitination of RET but not ΔTK RET. Expression vectors encoding HA-ATF4, Myc-RET mutants and FLAG-ubiquitin were transfected into U-2 OS cells as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody, and polyubiquitinated RET was detected by immunoblotting with anti-FLAG antibodies (upper panel). Expression level of each protein was assessed by anti-Myc, anti-HA, and anti-GAPDH antibodies (lower panel). B, Detection of the interaction between ATF4 and RET in vitro. Glutathione-agarose beads containing GST or GST-ATF4 were incubated with whole-cell extracts derived from U-2 OS cells expressing WT RET or ΔTK, C634W, K758M and M918T mutants. C, U-2 OS cells were transiently transfected with expression vectors encoding WT ATF4, WT RET, and ΔTK RET. After 24 h, the cell lysates were analysed by western blotting using antibodies against the indicated epitope tags. D and E, Coimmunoprecipitation of FLAG-ATF4 and HA-Cbl-c in U-2 OS/BTZ cells treated with 20 μM MG132 for 4 h. Overexpression of ATF4 increased the binding between RET and HA-Cbl-c (D), whereas knockdown of ATF4 decreased their binding (E). F and G, U-2 OS cells were cotransfected with HA-Cbl-c, Myc-RET, and FLAG-ubiquitin, and His-ATF4 (F) or siATF4 (G), and protein extracts were immunoprecipitated. Ubiquitination of RET was measured with an anti-Myc antibody. Cell lysates were immunoblotted with the indicated antibodies. H, U-2 OS and U-2 OS/BTZ cells transfected with increasing concentrations of the FLAG-ATF4 expression vector for 24 h were immunoblotted for endogenous proteins with the indicated antibodies. I, Expression of HSPA5 and CBLC in control vector- and FLAG-ATF4-transfected OS and OS/BTZ cells analysed by qRT-PCR. J, ATF4 binds to the promoter. U-2 OS and U-2 OS/BTZ cells were transfected with ATF4 or control vector. Chromatin immunoprecipitation was performed using either a control IgG antibody or an antibody against ATF4. PCR primers were designed to amplify the specific CBLC promoter fragment spanning from -382 to +1. Primers for the DDIT3 promoter were used as a positive control. Bars represent the mean ± SD, *P < 0.05.