Galactosyltransferase activity using the purified AtGALT29A/AtGALT31A complex in vitro . Microsomes were prepared from N. benthamiana leaves after expression of P19 only, AtGALT31A-GFP, HA-AtGALT29A or co-expression of HA-AtGALT29A and AtGALT31A-GFP, and subjected to immunoprecipitation using anti-GFP- or anti-HA-antibody. The conditions are indicated in the table at the bottom of (B). The immunoprecipitated samples were analyzed by the Western blot (A) and by the enzyme activity (B). A: The Western blot of P19, AtGALT31A-GFP, HA-AtGALT29A and AtGALT29A/AtGALT31A immunoprecipitated using GFP antibody. The result indicates co-purification of AtGALT31A-GFP (lane 5, indicated by the arrow at ca. 70 kDa) by immunoprecipitation of HA-AtGALT29A using anti-HA-antibody-agarose. The 50 kDa band detected in the lanes 3-5 is the heavy chain of HA antibody used for the immunoprecipitation, which is recognized by the secondary antibody used in the Western blot. B: Galactosyltransferase activity towards SP32-GFP and β-1,3-galactan acceptors. Affinity purified materials from the expression of P19 only, AtGALT31A-GFP, HA-AtGALT29A, or co-expression of HA-AtGALT29A and AtGALT31A-GFP using anti-GFP- or anti-HA-antibody were tested for enzyme activity using UDP-14[C]-Gal as substrate and SP32-GFP (lanes 1-5) or β-1,3-galactan (lanes 8-10) as acceptor, (n = 4). Control samples after co-expression of AtGALT31A-GFP or HA-AtGALT29A with HA-AtGLCAT14A (lane 6 and 7) were immunoprecipitated in the same way as for other samples and tested for the enzyme activity using UDP-14[C]-Gal as substrate and SP32-GFP as acceptor (lanes 6-7), (n = 3). These combinations are not suggested to form protein complexes based on the FRET analysis. Error bars showed standard deviations.