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. 2004 Sep 9;23(21):4232–4242. doi: 10.1038/sj.emboj.7600326

Figure 2.

Figure 2

Co-purification of TFIIS with Med13 and Cdk8 in cell-free extracts. (A) Co-purification of TFIIS∷3HA with immunoprecipitated Med13∷13Myc. Strains YPH499 (SPT8+) and YBV61 (spt8Δ) were transformed with plasmids encoding the Med13∷13Myc fusion (pVV226) and/or the TFIIS∷3HA fusion (pVV227). Protein crude extracts were prepared as described (Van Mullem et al, 2002b). Med13∷13Myc was immunoprecipitated by mouse monoclonal anti-Myc antibodies. Proteins (IP) were separated by SDS–PAGE, along with 10 μg of crude extracts, and revealed by Western blotting with monoclonal anti-Myc or anti-HA antibodies. The band noted IgG corresponds to the heavy chains of the mouse anti-Myc antibodies used for the immunopurification. (B) Co-purification of Med13∷3HA with immunoprecipitated TFIIS∷13Myc. Strain YPH499 was transformed with plasmids encoding the TFIIS∷13Myc fusion (pVV234) and/or the Med13∷3HA fusion (pVV229). Protein extraction, immunopurification and detection were as above. (C) Co-purification of Cdk8∷13Myc with immunoprecipitated TFIIS∷3HA. Strain YPH499 was transformed with plasmids encoding the TFIIS∷3HA fusion (pVV228) and/or the Cdk8∷13Myc (pVV233). TFIIS∷3HA was immunoprecipitated by mouse monoclonal anti-HA antibodies. Protein extraction and detection were as above. (D) Co-purification of TFIIS∷3HA with immunoprecipitated Cdk8∷13Myc. Strains YPH499 (MED13+) and YBV39 (med13Δ) were transformed with plasmids encoding the Cdk8∷13Myc (pVV233) and/or the TFIIS∷3HA fusion (pVV228). Protein extraction, immunopurification and detection were as in (A, B). No co-purification was observed when Cdk8∷13Myc and TFIIS∷3HA were expressed from their respective chromosomal locus (data not shown).