S6K1-2 | Ribosomal-protein S6 kinase homolog 1,2 - phosphorylated

Application example

30 µg of total protein of Arabidopsis thaliana 10 days old seedlings extracted with Lacus buffer with or without phosphatase inhibitors and kept at 4°C prior to denaturation for 5 min. at 95°C. Samples were separated on 6.5 % SDS-PAGE and blotted 2h to PVDF. Phosphatase treatment was done with CIAP ("+"  phosphatase inhibitors added, "-" phosphatase inhibitors not included). Blots were blocked with 5% milk in TBST for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for overnight at 4C with agitation. The antibody solution was decanted and the blot was washed three times for 15 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 1:10 000 in 2.5% milk in TBST for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was few minutes.

Courtesy of Dr. Rossana Henriques, CRAG, Spain

Ribosomal S6 kinase 1/2 (S6K1/2) involved in TOR signaling pathway, in osmotic stress response. Activated by PDK1 and repressed during osmotic stress. Expressed in all tissues, especially during high metabolic activity in growing buds, root tips, leaf margins and germinating seeds. Alternative names: AtPK1/AtPK6 S6K1), AtPK2/AtPK19 (S6K2).