Towbin buffer

Protein extracts were diluted in 5X SDS-PAGE loading buffer then boiled at 95°C for 10 min. Samples were subjected to SDS-PAGE electrophoresis at 125 V for approximately 1.5 h. Separated proteins were transferred to nitrocellulose membranes in either 1X transfer buffer (BioRad; S4 Table) at 1300 mA at 25°C, or in Towbin Buffer (BioRad; S4 Table) at 100 V at 4°C for 100 min. Membranes were blocked in Odyssey Blocking Buffer (LI-COR; S4 Table) for 1 h at 25°C. Membranes were blotted with primary antibody overnight at 4°C, with actin serving as a loading control unless otherwise stated. After three, 5 min washes with PBS-T (PBS, 0.1% Tween; S4 Table), membranes were incubated in secondary antibody conjugated to an IRDye (LI-COR; S4 Table) for 1 h followed by two, 5 min washes in PBS-T and one 5 min PBS wash. Membranes were then imaged with an Odyssey Fc Imager (LI-COR; S4 Table).

Protein extracts were diluted in 5X SDS-PAGE loading buffer then boiled at 95°C for 10 min. Samples were subjected to SDS-PAGE electrophoresis at 125 V for approximately 1.5 h. Separated proteins were transferred to nitrocellulose membranes in either 1X transfer buffer (BioRad; Table S4) at 1300 mA at 25°C, or in Towbin Buffer (BioRad; Table S4) at 100 V at 4°C for 100 min. Membranes were blocked in Odyssey Blocking Buffer (LI-COR; Table S4) for 1 h at 25°C. Membranes were blotted with primary antibody overnight at 4°C, with actin serving as a loading control unless otherwise stated. After three, 5 min washes with PBS-T (PBS, 0.1% Tween; Table S4), membranes were incubated in secondary antibody conjugated to an IRDye (LI-COR; Table S4) for 1 h followed by two, 5 min washes in PBS-T and one 5 min PBS wash. Membranes were then imaged with an Odyssey Fc Imager (LI-COR; Table S4).

Proteins were extracted from the inflorescences as described above. After protein extracts were quantified using the Bradford assay kit and 30 μg of total protein from each sample was separated on a 12.5 % SDS-PAGE gel (we use the acrylamide/bis solution (37.5:1, 2.6 % C), Carl Roth), proteins were transferred onto a PVDF membrane in the Towbin buffer with a wet blotting system (Bio-Rad), the membrane was then blocked with 5 %(w/v) non-fat dry milk in TBST (20 mM Tris-Cl, pH7.6, 137 mM NaCl, 0.1 %(v/v) Tween 20). To detect CDKA;1 proteins, the membrane was probed with a 1:5000 dilution of anti-PSTAIR monoclonal antibody (Sigma) and 1:10,000 HRP-conjugated anti-mouse antibody (KPL) in TBST. Enhanced chemiluminescence detection was performed with HRP substrate (Millipore). The signals were obtained by exposing a X-ray film.