Anti-14-3-3 GRF | General regulatory element

Brassica sp., Chlamydomonas reinhardtii, Glycine max,  Gossypium hirsutum, Nicotiana tabacum, Physcomitrium patens, Pisum sativum, Solanum tuberosum, Triticum aestivum

Left panel: 150 ng of recombinant Arabidopsis thaliana, Physcomitrella patens and Chlamydomonas reinhardtii GRFs (see more details below)  were separated on 12 % SDS-PAGE and blotted 1h to PVDF by semi dry blotting (0.8 mA/cm2 for 60 min). Blots were blocked with 2% skimmed milk powder dissolved in TBST (0.1 % Tween 20) for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in blocking solution and incubated for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:20 000 in  for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL Prime western blotting detection reagent according to the manufacturer's instructions. Exposure time was 10 minutes.

Right panel: 50 µg of a total cell extract of Arabidopsis thaliana wilde type. Conditions as above, exposure time 30 seconds.

GRF protein designations: At 14-3-3 lambda (GRF6), At 14-3-3 chi (GRF1), At14-3-3 psi (GRF3), At14-3-3 omega (GRF2), At14-3-3 upsilon (GRF5), At14-3-3 omicron (GRF11), At14-3-3 kappa (GRF8), Pp 14-3-3 Pp1s 73_133V6 (closest homolog to At GRF6), Cr 14-3-3 Cre 12.g559250 (closest homolog to AtGRF6).  Note that all recombinant GRFs were detected by this antibody, At14-3-3 kappa (GRF8) to a lesser extent.

Courtesy of Dr. Bernhard Wurzinger, University of Vienna, Austria

GRF (General regulatory factor 2) or 14.3.3s are 30 kDa proteins involved in protein interactions with target proteins containing phosphorylated target sites. Functions of 14.3.3s include acting as adaptors or scaffolds, stimulating protein-protein interaction, altering target protein activity, causing conformational changes of target proteins, regulating subcellular localisation and also facilitating transport (for example nuclear import/export and transport in the endomembrane system). A variety of apparently unrelated biological activities, including a role in development and signal transduction - brassinosteroid mediated signaling pathway.