S6K1/2 | Ribosomal S6 kinase 1/2

AS12 1855 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana

Product Information

Immunogen

KLH-conjugated peptide, derived from Arabidopsis thaliana S6K1: UniProt: P42818, TAIR: AT3G08730 and S6K2: UniProt: Q39030, TAIR: AT3G08720. Due to high amino acid homology, chosen peptide is conserved in both proteins: S6K1 and S6K2.

Host Rabbit

Clonality Polyclonal

Purity Immunogen affinity purified serum in PBS pH 7.4. Contains 0.02% sodium azide.

Format Lyophilized

Quantity 50 µg

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles,Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.

Tested applications Western blot (WB)

Recommended dilution 1 : 750-1 : 1000 (WB)

Expected | apparent MW 52,6 kDa (S6K1) and 53 kDa (S6K2)

Reactivity

Confirmed reactivity Arabidopsis thaliana

Predicted reactivity Thelungiella halophila
Species of your interest not listed? Contact us

Not reactive in Setaria viridis, Solanum lycopersicum; Vitis vinifera

Application examples

Application examples application example

western blot using anti-S6K/2 antibodies

20 µg of total protein from flowers and leaves of transgenic Arabidopsis thaliana lines were analysed (expressing the genomic copy of S6K1 tagged with FLAG epitop under the control of its own promoter) extracted with homogenization buffer were separated on 10% SDS-PAGE and blotted 2h to PVDF. 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 1:750 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

western blot using anti-S6K polyclonal antibodies

western blot using anti-S6K polyclonal antibodies

20 µg of total protein from Arabidopsis thaliana total wilde type (Col-0), deletion mutant (s6k1-1), overexpression mutant (S6K1g-CFP) extracted with homogenization buffer were separated on 10% SDS-PAGE and blotted 2h to PVDF. 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 1:1000 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

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 29079776

Journal: Nat Commun

Figure Number: 2G

Published Date: 2017-10-27

First Author: Dong, Y., Silbermann, M., et al.

Impact Factor: 13.783

Open Publication

Specific sensing of C/N- or S-precursors for Cys results in distinct transcriptome reprogramming and specific translation regulation. a Venn diagrams and b–e functional category analysis (p?<?0.05, FDR?<?0.25) for transcriptional response in roots of 7-week-old serat tko and sir1-1 plants. The compared sets of genes contained transcripts, which were significantly (p?<?0.05) up- or downregulated by more than 1.25-fold in sir1-1 or serat tko in comparison to the wild type. The details of the highlighted categories with underlines are further presented in Supplementary Figs. 2 and 4. f Specific downregulation of 18s rRNA in response to sulfide limitation (n?=?3, mean?±?s.e.m., one-way ANOVA, *p?<?0.05). g TOR and GCN2 activity was determined by antibodies against S6K-p, S6K (apparent sizes: 52?kDa) and eIF2?-p (apparent size: 43?kDa) and h calculated by the ratio of S6K-p/S6K and eIF2?-p/LC in x-fold of wild type (n?=?3, mean?±?s.e.m., one-way ANOVA, *p?<?0.05)

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 29079776

Journal: Nat Commun

Figure Number: 3A

Published Date: 2017-10-27

First Author: Dong, Y., Silbermann, M., et al.

Impact Factor: 13.783

Open Publication

Sulfur availability downregulates TOR activity and sugar metabolism. a Immunological detection of S6K-p, S6K (52?kDa) and eIF2?-p (43?kDa) with specific antisera was used for calculation of relative TOR activity by the ratio of S6K-p/S6K in x-fold of wild type (n?=?3, mean?±?s.e.m., t-test, *p?<?0.05). b 18s and 25s rRNA level were measured in the shoot of wild type under sulfur deficiency (n?=?3, mean?±?s.e.m., t-test, *p?<?0.05). c Growth phenotype of the des1-1 mutant having low sulfide level in the cytosol. Scale bar, 2?cm. d Immunological detection of S6K phosphorylation status in the genotypes shown in c (apparent sizes of S6K-p, S6K: 52?kDa). e Calculation of TOR activity by the ratio of S6K-p/S6K in x-fold of wild type (n?=?3, mean?±?s.e.m., one-way ANOVA, *p?<?0.05). f, g Functional category analysis (p?<?0.05, FDR?<?0.25) for downregulated genes in shoots of 7-week-old sir1-1 and sulfur-deficient WT plants. The compared sets of genes contained transcripts, which were identified by microarray analysis to be significantly (p?<?0.05) downregulated by more than 1.25-fold. The categories related to sugar metabolism are highlighted with underlines. h Soluble sugar levels in shoots of 7-week-old sulfur-deficient plants (n?=?4, mean?±?s.e.m., t-test, *p?<?0.05)

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 29079776

Journal: Nat Commun

Figure Number: 3D

Published Date: 2017-10-27

First Author: Dong, Y., Silbermann, M., et al.

Impact Factor: 13.783

Open Publication

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 29079776

Journal: Nat Commun

Figure Number: 4B

Published Date: 2017-10-27

First Author: Dong, Y., Silbermann, M., et al.

Impact Factor: 13.783

Open Publication

Sulfur availability regulates root meristem and plant growth via glucose-TOR. a Phenotype of chimeric plants obtained by grafting of wild-type (WT) and sir1-1 organs. Identity of organs is depicted as shoot/root. Scale bar, 2?cm. b–e Feeding of serat tko and sir1-1 seedlings with glucose. b Immunological detection of S6K-p, S6K (apparent sizes: 52?kDa), and eIF2?-p (apparent size: 43?kDa) with specific antisera in wild-type serat tko and sir1-1. c, d Phenotype of wild-type serat tko and sir1-1 plants in the absence and presence of glucose. Scale bar, 1?cm. e Impact of glucose on root meristem activity in wild-type serat tko and sir1-1. Scale bar, 25?µm. f Root meristem activity of 7-day-old WT seedlings treated with TOR inhibitor (AZD-8055) or GCN2 activator (Chlorosulfuron, CHL) for 2?h. Scale bar, 25?µm

Additional information

Additional information So far in applied conditions and extracts endogenous S6K1/2 protein is detectable as a very weak band

Keeping the samples at 4°C all times is of crucial importance, Laucs buffer was used in example below since this is the one which was used routinely by this test laboratory for the work with kinases, Some unspecific bands can be seen, depending upon western blot protocol which is used, Therefore please consider to use a negative control together with your samples

Related products

AS12 2608 | Anti-TOR | Target of rapamycin, rabbit antibodies
AS13 2664 | Anti-S6K1-2 | Ribosomal-protein S6 kinase homolog 1,2 - phosphorylated, rabbit antibodies

Plant protein extraction buffer

Secondary antibodies

Background

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).

Product citations

Selected references González-López et al. (2021). Growth promotion in Arabidopsis thaliana by bacterial cyclodipeptides involves the TOR/S6K pathway activation. Journal of Plant Physiology. Volume 257, 2021, 153343,ISSN 0176-1617, https://doi.org/10.1016/j.jplph.2020.153343.
Salazar-Diaz et al. (2021) TOR senses and regulates spermidine metabolism during seedling establishment and growth in maize and Arabidopsis. iScience. 2021 Oct 12;24(11):103260. doi: 10.1016/j.isci.2021.103260. PMID: 34765910; PMCID: PMC8571727.
Angelos & Brandizzi (2021). The UPR regulator IRE1 promotes balanced organ development by restricting TOR-dependent control of cellular differentiation in Arabidopsis. Plant J. 2021 Dec 11. doi: 10.1111/tpj.15629. Epub ahead of print. PMID: 34902186.
Kazibwe et al. (2020). TOR mediates the autophagy response to altered nucleotide homeostasis in a ribonuclease mutant. J Exp Bot. 2020 Sep 9;eraa410.doi: 10.1093/jxb/eraa410.
Dong et al. (2019). The Arabidopsis THADA homologue modulates TOR activity and cold acclimation. Plant Biol (Stuttg). 2019 Jan;21 Suppl 1:77-83. doi: 10.1111/plb.12893.
Inaba and Nagy (2018). Tombusvirus RNA replication depends on the TOR pathway in yeast and plants. Virology. 2018 Jun;519:207-222. doi: 10.1016/j.virol.2018.04.010.
Pfeiffer et al. (2016). Integration of light and metabolic signals for stem cell activation at the shoot apical meristem. Elife. 2016 Jul 11;5. pii: e17023. doi: 10.7554/eLife.17023.