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. Author manuscript; available in PMC: 2011 May 11.
Published in final edited form as: Oncogene. 2010 Sep 6;29(45):6004–6015. doi: 10.1038/onc.2010.336

Figure 2.

Figure 2

C/EBPbeta1 is protealyzed by the proteasome in cells in which the Ras pathway is activated a. MCF10A-Ras cells infected with LZRS-T7-C/EBPbeta1-IRES-eGFP and sorted by FACS for GFP positive cells. This immunoblot is three weeks post-sorting. Lane 1 is uninfected MCF10A-Ras cells, lane 2 is MCF10A-Ras-C/EBPbeta1 cells, lane 3 is MCF10A-Ras-C/EBPbeta1 treated with 50uM MG132 (re-suspended in DMSO; Calbiochem) for 4 hours, and lane 4 is MCF10A-Ras-C/EBPbeta1 cells treated with 50uM MG132 for 8 hours. Equal amounts of total protein were loaded in each lane of a 10% SDS-PAGE. The Santa Cruz C-19 C-terminal C/EBPbeta antibody was used for immunoblot analysis at 1:5000. Arrows indicate the different C/EBPbeta isoforms. b. Cell lysates were prepared from MCF10A-Ras cells untreated (lane 1) or treated with 50uM MG132 for 8 hours (lane 2). Equal amounts of total protein were loaded into each lane of a 10% SDS-PAGE. An antibody specific for the N-terminal 21 amino acids specific to C/EBPbeta1 (Abcam 18F8) was used for immunoblot analysis (1:2000). c. MDA231 breast cancer cells infected with LZRS-T7-C/EBPbeta1-IRES-eGFP and sorted by FACs for GFP positive cells. Lane 1 is uninfected MDA231 cells, lane 2 is uninfected MDA231 cells treated with 50uM MG132, lane 3 is MDA231-C/EBPbeta1, and lane 4 is MDA231-C/EBPbeta1 cells treated with 50uM MG132 for 8 hours. Equal amounts of total protein were loaded in each lane of a 10% SDS-PAGE. Immunoblot analysis was performed with a T7 tag mouse monoclonal antibody (Novagen) at 1:10000. The arrow indicates p52-T7-C/EBPbeta1. Beta-tubulin was used as the loading control for all of the above immunoblots (Sigma T7816). d. MCF10A-Ras cells infected with LZRS-T7-C/EBPbeta1-IRES-eGFP and sorted by FACS for GFP positive cells. Lane 3 is uninfected MCF10A-Ras cells. Cells were untreated (lanes 1, 3 and 4) or treated with 50uM MG132 for 8 hours and 5mM N-ethylmaleimide for 30 minutes (lanes 2 and 5). Lanes 1 and 2 are cell lysates whereas lanes 3-5 are immunoprecipitations with T7-tag antibody beads (Novagen). Immunoprecipitations were performed as described previously (Eaton and Sealy, 2003) with the following exceptions: the immunoprecipitations were for 15 minutes and 50uM MG132 and 5mM N-ethylmaleimide were included in the immunoprecipitation buffer. Immunoblot analysis was performed with the Santa Cruz C-19 C-terminal C/EBPbeta antibody (lanes 1 and 2) or anti-Ubiquitin antibody (Enzo Life Sciences FK2). (10A = MCF10A, beta = C/EBPbeta, 231 = MDA231, MG = MG132)