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. 2016 Nov 8;291(52):26922–26933. doi: 10.1074/jbc.M116.752063

FIGURE 2.

FIGURE 2.

The EWS-FLI1 protein displays a high turnover. A, scheme illustrating GPS approach in which a reporter construct, DsRed-IRES-EGFP, is fused at the C terminus of EGFP to a protein of interest. The EGFP/DsRed ratio is determined by FACS and represents a measure for protein stability. B, EWS-FLI1 turnover measured by GPS. HEK293T were transduced for 72 h with a reporter construct fused to EWS-FLI1 or degron (d) motifs with half-lives of 1 (d1h), 4 (d4h), and 24 h (d24h) and analyzed by FACS. The reporter construct fused to EWS-FLI1 was additionally incubated with 20 μm MG-132 or 20 μg/ml CHX for 8 h or with 50 nm LMB for 4 and 8 h (dotted line) (C). D, EWS-FLI1 stability in Ewing cell line A673. GPS analysis of reporter constructs fused to EWS-FLI1 or standard degron motifs 72 h after transduction. E, A673 cells stably transfected with reporter-EWS-FLI1 construct were sorted and incubated with DMSO, 20 μm MG-132, or 20 μg/ml CHX for 8 h. d1h, d4h, and d24h, degron (d) motifs with half-lives of 1, 4, and 24 h, respectively.