total s6k  (Santa Cruz Biotechnology)

Journal: Journal of Pharmaceutical Analysis

Article Title: Hemodynamic disturbance and mTORC1 activation: Unveiling the biomechanical pathogenesis of thoracic aortic aneurysms in Marfan syndrome

doi: 10.1016/j.jpha.2024.101120

Figure Lengend Snippet: Mechanistic target of rapamycin complex 1 (mTORC1) pathway intensification in Fbn1 + /C1039G mouse aortas over time. (A) Morphology of ascending aortas at different growth stages. (B) Ultrasound showing aortic dilation (end-systolic diameter of the aorta) of Fbn1 + /C1039G mice at 8, 12, and 16 week. (C) The analysis of aortic root diameter tracking by ultrasound of Fbn1 + /+ wild-type (WT) and Fbn1 + /C1039G mice. (D) 3D reconstructions of aortic progression over time. (E) Computational fluid dynamic (CFD) analyses indicate increased disturbed flow correlating with aortic expansion. (F) Western blots and the relative protein level of phosphorylated S6 (p-S6), total S6 (t-S6), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the WT and Fbn1 + /C1039G mice at 8, 12, and 16 week. (G) Western blots and the relative protein level of p-S6, t-S6, and GAPDH of the WT, Fbn1 + /C1039G at 20 and 24 week. (H) Western blots and the relative protein level of phosphorylated S6 kinase (p-S6K), t-S6K, p-S6, t-S6, and β-actin of the WT and Fbn1 + /C1039G mice at 30 week. Data are presented as mean ± standard error of the mean (SEM) for n = 6 per group. Statistical significance was determined by one-way analysis of variance (ANOVA), with ∗ P < 0.05, and ∗∗ P < 0.01, indicating significant differences; NS: not significant.

Article Snippet: Antibodies against phosphorylated ribosomal protein S6 kinase (p-S6K, Thr421/Ser 424, 9204), total S6K (t-S6K, 9202), p-S6 (Ser 235/236, 4858), t-S6 (2217), phosphorylated extracellular signal-regulated kinase ( p -ERK, 9101), and p -Smad 2 (18338) were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot

Journal: Journal of Pharmaceutical Analysis

Article Title: Hemodynamic disturbance and mTORC1 activation: Unveiling the biomechanical pathogenesis of thoracic aortic aneurysms in Marfan syndrome

doi: 10.1016/j.jpha.2024.101120

Figure Lengend Snippet: Rapamycin mitigates mechanistic target of rapamycin complex 1 (mTORC1) activation induced by oscillatory shear stress (OSS) in Fbn1 +/C1039G Mice. (A) Visualization of blood flow velocity and streamlines within the aortic lumen captures the dynamic hemodynamic alterations related to aneurysm progression and the disturbed flow of the three groups: wild-type (WT) littermates, Fbn1 +/C1039G mice, and Fbn1 +/C1039G mice subjected to long-term rapamycin treatment ( Fbn1 +/C1039G + RA). (B) The presence of OSS across the aortic regions of the three groups: WT littermates, Fbn1 +/C1039G mice, and Fbn1 +/C1039G + RA, with OSS areas marked in gray. (C) Comparative aortic velocity mapping and cross-sectional OSS distribution among the three groups, with OSS zones delineated by white dashed lines. (D) Immunohistochemical detection of phosphorylated S6 (p-S6) in ascending aortic sections from the three groups. (E) Enlarged view of the OSS regions from the respective groups. (F) Quantification of p-S6 fluorescence intensity across the groups, indicating the modulation of mTORC1 signaling by rapamycin. (G–I) Western blot analysis profiling the expression of phosphorylated S6 kinase (p-S6K), total S6 kinase (t-S6K), phosphorylated S6 (p-S6), total S6 (t-S6), and statistical evaluation of protein expressions at 12 (G), 20 (H), and 30 week (I). Data expressed as mean ± standard error of the mean (SEM) for n = 6 per group. One-way analysis of variance (ANOVA) was utilized to assess statistical significance, denoted by ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001; NS: not significant.

Article Snippet: Antibodies against phosphorylated ribosomal protein S6 kinase (p-S6K, Thr421/Ser 424, 9204), total S6K (t-S6K, 9202), p-S6 (Ser 235/236, 4858), t-S6 (2217), phosphorylated extracellular signal-regulated kinase ( p -ERK, 9101), and p -Smad 2 (18338) were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Activation Assay, Shear, Immunohistochemical staining, Fluorescence, Western Blot, Expressing

Journal: Plant Physiology

Article Title: 40S Ribosomal protein S6 kinase integrates daylength perception and growth regulation in Arabidopsis thaliana

doi: 10.1093/plphys/kiae254

Figure Lengend Snippet: S6K1 and S6K2 expression and protein levels are regulated by photoperiod and the circadian clock. S6K1 and S6K2 transcript levels were detected by RT-qPCR in 2-week-old wild type seedlings grown under short day (SD, A) , long day (LD, A) and continuous light conditions (LL, B) . Results were analyzed in technical triplicates and normalized to Actin 2 in two independent biological replicates in (A) , in (B) the results of one representative biological replicate are shown. Error bars refer to ± standard deviation values. S6K total protein levels determined in 10 day-old seedlings grown under either short day (SD) (C) or long day (LD) conditions (E) . In parallel, the levels of phosphorylated S6K protein (S6K-P) were detected in the same samples, in SD (D) and in LD (F) . Loading control corresponds to Coomassie Brilliant Blue staining of the same western membranes used either for anti-S6K or anti-S6K-P detection. Arrows point to S6K or S6K-phosphorylated bands (S6K-P), star indicates an unspecific band. Underneath each western blot is shown the average signal intensity of either the S6K or S6K-P signals after normalization with loading control in two independent biological replicates. Error bars represent ± standard error values. MW represents protein molecular weight marker bands in KDa. Time (h) refers to hours after lights on. Dark gray rectangles correspond to the dark period under LD, whereas lighter gray rectangles correspond to the dark period under SD. Dotted rectangles refer to the subjective night period.

Article Snippet: Anti-total S6K (Agrisera Antibodies, AS12 1855) and anti-phosphorylated S6K antibodies (Agrisera Antibodies, AS13 2664) were used in a 1:1,000 or 1:750 dilution respectively, in a 7.5% milk TBS-T solution.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Control, Staining, Western Blot, Molecular Weight, Marker

Journal: Plant Physiology

Article Title: 40S Ribosomal protein S6 kinase integrates daylength perception and growth regulation in Arabidopsis thaliana

doi: 10.1093/plphys/kiae254

Figure Lengend Snippet: S6K total protein levels are regulated by the 26S proteasome. A) S6K total protein levels present in 10 day-old seedlings grown under SDs conditions, either mock-treated (DMSO—Dimethyl sulfoxide; left panels) or incubated with the 26S proteasome inhibitor (MG132; right panels). Seedlings were incubated at ZT3 for the times described (h). The upper panels show total S6K levels, whereas the lower panels reflect the actin levels. Asterisk (*) refers to an unspecific band. MW refers to protein molecular weight marker bands shown in KDa. B) Plotted averages of S6K specific band in two independent biological duplicate experiments under the exact same conditions as described in (A) . Error bars represent ± standard error values. Details for quantification of results are provided in the Materials and methods section. Statistically significant differences (* indicates P < 0.05) were calculated using Student's t test for each time point analysed.

Article Snippet: Anti-total S6K (Agrisera Antibodies, AS12 1855) and anti-phosphorylated S6K antibodies (Agrisera Antibodies, AS13 2664) were used in a 1:1,000 or 1:750 dilution respectively, in a 7.5% milk TBS-T solution.

Techniques: Incubation, Molecular Weight, Marker

Journal: Plant Physiology

Article Title: 40S Ribosomal protein S6 kinase integrates daylength perception and growth regulation in Arabidopsis thaliana

doi: 10.1093/plphys/kiae254

Figure Lengend Snippet: The circadian F-box protein ZTL modulates S6K protein levels and half-life. Total S6K protein accumulates at higher levels and for a longer period of time in ztl-3 mutant plants than in wild type (WT, Col-0) seedlings grown under SD conditions. A) Total S6K and actin protein levels in WT plants (upper panels) and ztl-3 mutants (lower panels) were detected by western blot analysis using anti-S6K and anti-actin specific antibodies. Arrows point to S6K and actin bands, respectively. Star indicates an unspecific band. B) Average S6K signal intensity after normalizing with the corresponding actin signal in three independent biological replicates. C) Similarly, phosphorylated S6K is present for a longer period of time in the ztl-3 mutants (lower panels) when compared with WT (upper panels) seedlings. Loading control was determined by actin levels in the same blots. Arrows point to S6K-P and actin bands, respectively. Star indicates an unspecific band. D) Average signal intensity for S6K-P after normalizing with actin loading control in two independent biological replicates. E) Upper panels: SD grown 10 day-old WT and ztl-3 seedlings were incubated at ZT3 with the protein synthesis inhibitor cycloheximide (CHX) and maintained under white light for the indicated hours (h). The s6k1.1 mutant was used as negative control. Shown is one representative experiment out of two biological replicates. Arrows point to S6K and actin bands, respectively. Star indicates an unspecific band. Lower panel: average S6K signal intensity after normalizing with the loading control in two independent biological replicates. F) Upper panels: SD grown 10 day-old WT and ztl-3 seedlings were incubated at ZT3 with cycloheximide (CHX) and maintained in the dark for the indicated hours (h). Shown one representative experiment out of two biological replicates. Arrows indicate S6K or actin levels, asterisk points to unspecific band. Lower panel: Quantification of the average S6K signal after normalization with the loading control (actin) in two independent biological replicates. Gray rectangle represents the incubation in the dark. Error bars correspond to ± standard error values. MW represents protein molecular weight marker bands shown in KDa. Time refers to hours (h) after lights on; white and gray rectangles indicate the light and dark periods, respectively. Statistically significant differences (* P < 0.05, *** P < 0.001) were calculated using Student's t test for each time point analyzed.

Article Snippet: Anti-total S6K (Agrisera Antibodies, AS12 1855) and anti-phosphorylated S6K antibodies (Agrisera Antibodies, AS13 2664) were used in a 1:1,000 or 1:750 dilution respectively, in a 7.5% milk TBS-T solution.

Techniques: Mutagenesis, Western Blot, Control, Incubation, Negative Control, Molecular Weight, Marker

Journal: Plant Physiology

Article Title: 40S Ribosomal protein S6 kinase integrates daylength perception and growth regulation in Arabidopsis thaliana

doi: 10.1093/plphys/kiae254

Figure Lengend Snippet: Regulation of S6K1 levels under SD conditions is required for efficient use of resources and adequate growth responses. A, B) Variable S6K1 levels affect diel accumulation of sugars. Top to bottom: accumulation of Starch, Sucrose, Glucose, and Fructose in 30-day old soil-grown seedlings under SD conditions. Pools of three rosettes were analyzed in five biological replicates in Col-0 and s6k1.1 mutants (A) and in three biological replicates in Col-0 and S6K1p::S6K1g-CFP#11.6 overexpression line (B) . The results are the mean ± SD. Statistically significant differences are indicated by asterisks (Student's t -test): * P < 0.05, ** P < 0.01, and *** P < 0.001. FW refers to fresh weight of analyzed plants (g). C) Current working model describing overall impact of circadian (clock drawing) and photoperiod (sun drawing) regulation of S6K1 and consequently, TOR pathway activity. S6K1 protein levels start to increase at dawn and will be maximal between ZT3-6 when TOR-dependent phosphorylated S6K also accumulates. At dusk, ZTL will mediate S6K1 proteasome-dependent degradation. S6K1 protein levels will then be minimal during the long dark period. This regulatory network will allow the coordination between C/energy use and TOR pathway activity (depicted as translational capacity) to ensure that plant growth responses are perfectly matched with the available resources especially during the long night. White cubes show available C for growth and its pattern of accumulation is depicted by the yellow line, green rosettes represent Arabidopsis, and gray circles connected to the black lines refer to ribosomes associated to mRNAs (to depict translational capacity). White and gray rectangles indicate the light and dark periods, respectively.

Article Snippet: Anti-total S6K (Agrisera Antibodies, AS12 1855) and anti-phosphorylated S6K antibodies (Agrisera Antibodies, AS13 2664) were used in a 1:1,000 or 1:750 dilution respectively, in a 7.5% milk TBS-T solution.

Techniques: Starch, Over Expression, Activity Assay

Journal: Microbial cell factories

Article Title: The transcription factor GCN4 contributes to maintaining intracellular amino acid contents under nitrogen-limiting conditions in the mushroom Ganoderma lucidum.

doi: 10.1186/s12934-023-02213-z

Figure Lengend Snippet: Fig. 4 GCN4 inhibits TORC1 activity under low-nitrogen conditions. Western blot analysis of S6K phosphorylation levels in the WT and gcn4-silenced strains cultured under 3 mM and 60 mM Asn, with histograms showing the relative ratio of p-S6K/Total-S6K. The p-S6K/Total-S6K ratio in the WT strain grown under 60 mM Asn was defined as 1. The data are presented as the mean ± SD (n = 3). Statistical significance is represented by different letters ac cording to Tukey′s test (P < 0.05)

Article Snippet: Western blotting was conducted according to a previous method using polyclonal antibodies against GCN4 (1:1,000) and anti-total p70 S6K, anti-phospho (T389)S6K, and β-tubulin antibodies (9202, 9205, and 2146, 1:2,000, Cell Signaling Technology, Shanghai, China).

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Cell Culture