Fig. 1.

Specificity test of antibodies used for detection of the Medicago retinoblastoma-related protein (MsRBR) and its phosphorylated form (phospho-MsRBR) in cells at stationary or exponential growing phase. (A) Antigen competition assay for anti-AtRBR1 antibody: 1, western blot of total protein extract with anti-AtRBR1 antibody detected the MsRBR1 protein (115 kDa); 2, immunoblot of total protein extract with anti-AtRBR1 antibody pre-incubated with the purified (His)₆-tagged C-terminal part of the MsRBR1 protein failed to detect the MsRBR protein (115 kDa). (B) Functionality test of anti-human pRb phosphopeptide antibody by western blot of the in vitro phosphorylated recombinant C-terminal fragment of MsRBR protein after incubation with p13^SUC1-bound kinase complex. Upper panel, Ponceau S-stained filter used for immunoblot assay; middle panel, immunoblot with antibody produced against the phosphopeptide corresponding to residues around Ser807/811 of human pRb; lower panel, detection of incorporated [³²P]inorganic phosphate by Phosphor Imager SI (Molecular Dynamics). (C) Alfalfa cells at exponential phase have an increased amount of the MsRBR protein in comparison with cells at stationary phase. Lanes 1–3, protein extracts from 7-day-old cultures (stationary phase); lanes 4–6, protein extracts from 4-day-old cultures (exponential phase); 1, 4, control cultures; 2, 5, protein extracts treated with phosphatase buffer; 3, 6, protein extracts with calf intestinal alkaline phosphatase (CIAP). (D) Western blots with the anti-human pRb phosphopeptide antibody detected reduced amounts of phospho-MsRBR protein after phosphatase treatment and showed significantly higher amounts of phospho-MsRBR protein in cells at exponential growing phase. Lanes 1–3, protein extracts from 7-day-old cultures (stationary phase); lanes 4–6, protein extracts from 4-day-old cultures (exponential phase); 1, 4, control cultures; 2, 5, protein extracts treated with phosphatase buffer; 3, 6, protein extracts with CIAP.