Polysomal Profiling and Phospho-Protein Analysis

Polyribosomes were obtained from A. thaliana seedlings and analysed as described previously (42 (link)). Crude polysomal extracts were obtained from 200 mg of Arabidopsis seedlings treated or not with 1 μM auxin during 24 h and resolved on 7–47% sucrose gradient centrifuged for 3 h at 38 000 rpm (rotor SW41-Ti, Beckman Coulter). Analysis of polysome profiles was performed with an absorbance detector at 254 nm and sucrose gradients collected with a BioLogic Duoflow fractions collector (Biorad) into 18 fractions of 500 μl each. Samples from the light (LP), heavy polysome (HP) and non-polysomal 80S, 60S and 40S (NP) fractions were then separated on a 12.5% SDS-PAGE followed by immunoblotting to determine total and phosphorylated RPS6 and RISP protein levels. Rabbit polyclonal antibodies raised against RISP and eL13 were described previously (34 (link)) and (43 (link)), respectively. Polyclonal Phospho-(Ser/Thr)Akt substrate (RxRxxS/T) antibody for RISP-P detection was from Cell Signaling Technology. Antibodies against eS6 (RPS6) and phospho-specific antibodies against S240-P were generated by Agrisera, Umeå, Sweden and kindly provided by Albrecht G. von Arnim (University of Tennessee, Knoxville, USA). Polyclonal anti-eS14 was from Agrisera.

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Mancera-Martínez E., Dong Y., Makarian J., Srour O., Thiébeauld O., Jamsheer M., Chicher J., Hammann P., Schepetilnikov M, & Ryabova L.A. (2021). Phosphorylation of a reinitiation supporting protein, RISP, determines its function in translation reinitiation. Nucleic Acids Research, 49(12), 6908-6924.

Publication 2021

SW41-Ti

Antibodies Antibody Arabidopsis Auxin Biologic Crude extracts Light Phospho specific antibodies Polysomal Protein Rabbit Sds page Seedlings Sucrose

Corresponding Organization :

Other organizations : Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Centre National de la Recherche Scientifique

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Variable analysis

independent variables

Treatment with 1 μM auxin during 24 h

dependent variables

Total and phosphorylated RPS6 and RISP protein levels
Polysome profiles

control variables

Crude polysomal extracts obtained from 200 mg of Arabidopsis seedlings
Sucrose gradient centrifugation for 3 h at 38 000 rpm
Absorbance detection at 254 nm
Separation of samples on 12.5% SDS-PAGE
Immunoblotting to determine protein levels

positive controls

Rabbit polyclonal antibodies raised against RISP and eL13 described previously (34 and 43)

negative controls

Crude polysomal extracts obtained from Arabidopsis seedlings not treated with auxin

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