Fig. 1. hCCR4, hCAF1 and hPOP2 form common complexes in mammalian cells. (A) HeLa cell lysate was analyzed by gel filtration chromatography using a Superose 6 (HR10/30) column. Protein extracts were pre-cleared by centrifugation at 22,000 g for 20 minutes, then 300 µl of the sample (4 mg) were loaded onto the column. The flow rate was 0.4 ml/minute, and 250 µl were collected in each fraction, from which 50 µl were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot using anti-hCCR4, -hCAF1 and -hPOP2 antibodies. The numbers indicate fractions and the control lines represent extracts from HeLa cells transfected respectively with hCCR4^FLAG-, hCAF1^FLAG- and hPOP2^FLAG-expressing plasmids. (B) hCAF1 was associated with hCCR4 in high molecular weight complex. Cellular extracts from HeLa-expressing Flag-tagged CCR4 were fractionated by gel filtration chromatography using a Superose 6 column as described before. Fractions 6-9 were pooled and incubated with anti-Flag M2 affinity gel at 4°C for 8-12 hours. Bound proteins were eluted with the sample buffer and boiled. Western blots were performed using anti-hCCR4 and anti-hCAF1 antibodies. Molecular size markers are given in kilodaltons.