Proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose membranes and analysed with specific antibodies against HA (Roche), Flag (Sigma Aldrich), GST (Santa Cruz Biology), GFP (Roche), RFP (5F8, Chromotek), Myc (Clone 4A6, Merck), eIF4A (St. John’s laboratory), eIF3D (sc-28856, Santa Cruz), AteIF4E and AteIF(iso)4E (kindly provided by Dr. J. L. Gallois, INRA, France) and TaeIF4E (kindly provided by Dr. Karen Browning, University of Texas, USA).
The C-terminal part of CERES (base pairs 1471-1794) was cloned into pDEST17 vector (Invitrogen) and transformed into E. coli strain BL21. Cells were induced for protein expression by adding 0.1 mM IPTG and the proteins were separated by SDS-polyacrylamide gel electrophoresis. Following SDS-PAGE, the gels were incubated in cold 2 M KCl until the protein bands became opaque. The band corresponding to Ct-CERES was excised from the gel. This band was used to immunise rabbits for antiserum production by Pineda Antibody Services (Germany).
The C-terminal part of CERES (base pairs 1471-1794) was cloned into pDEST17 vector (Invitrogen) and transformed into E. coli strain BL21. Cells were induced for protein expression by adding 0.1 mM IPTG and the proteins were separated by SDS-polyacrylamide gel electrophoresis. Following SDS-PAGE, the gels were incubated in cold 2 M KCl until the protein bands became opaque. The band corresponding to Ct-CERES was excised from the gel. This band was used to immunise rabbits for antiserum production by Pineda Antibody Services (Germany).