Journal: Molecular Pain

Article Title: Activation of mammalian target of rapamycin mediates rat pain-related responses induced by BmK I, a sodium channel-specific modulator

doi: 10.1186/1744-8069-9-50

Figure Lengend Snippet: Immunoreactivity of p-mTOR in the spinal cord dorsal horn after intraplantar BmK I injection. Compared with the saline group (A , B) , BmK I treated groups (C - F) showed obvious immunoreactivity of p-mTOR in both ipsi- and contralateral spinal cord dorsal horn.

Article Snippet: The membranes were incubated with the following primary antibodies which were diluted in PBS-T containing 5% nonfat milk for 5 h at room temperature: rabbit polyclonal antibody against p-mTOR (Ser2448, 1:1000; #2971; Cell Signaling Technology, MA [ ])/rabbit monoclonal antibody against mTOR (7C10, 1:1200; #2983; Cell Signaling Technology, MA)/rabbit polyclonal antibody against p-p70 S6K (Thr421/Ser424, 1:1500; sc-7984-R; Santa Cruz, CA [ ])/rabbit monoclonal antibody against p-4E-BP1 (Thr37/46, 1:1000; #2855; Cell Signaling Technology, MA)/rabbit polyclonal antibody against Actin (I-19, 1:1000; sc-1616; Santa Cruz, CA).

Techniques: Injection, Saline

Journal: Molecular Pain

Article Title: Activation of mammalian target of rapamycin mediates rat pain-related responses induced by BmK I, a sodium channel-specific modulator

doi: 10.1186/1744-8069-9-50

Figure Lengend Snippet: BmK I-induced changes in spinal levels of p-mTOR, p-4E-BP1 and p-p70 S6K assessed by Western blot. (A) & (F) , representative western blots showing levels of p-mTOR, p-4E-BP, p-p70 S6K, total mTOR and β-actin in both ipsi- (A) and contralateral (B) side of the spinal cord. (B - E) & (G - J) , histograms represent the mean levels with respect to each control group at different time points after intraplantar BmK I injection. The data are presented as mean ± S.E.M. of three rats per group. * p <0.05, ** p <0.01, *** p <0.001, compared with control group by One-way ANOVA, followed by Bonferroni's post hoc test.

Article Snippet: The membranes were incubated with the following primary antibodies which were diluted in PBS-T containing 5% nonfat milk for 5 h at room temperature: rabbit polyclonal antibody against p-mTOR (Ser2448, 1:1000; #2971; Cell Signaling Technology, MA [ ])/rabbit monoclonal antibody against mTOR (7C10, 1:1200; #2983; Cell Signaling Technology, MA)/rabbit polyclonal antibody against p-p70 S6K (Thr421/Ser424, 1:1500; sc-7984-R; Santa Cruz, CA [ ])/rabbit monoclonal antibody against p-4E-BP1 (Thr37/46, 1:1000; #2855; Cell Signaling Technology, MA)/rabbit polyclonal antibody against Actin (I-19, 1:1000; sc-1616; Santa Cruz, CA).

Techniques: Western Blot, Control, Injection

Journal: Molecular Pain

Article Title: Activation of mammalian target of rapamycin mediates rat pain-related responses induced by BmK I, a sodium channel-specific modulator

doi: 10.1186/1744-8069-9-50

Figure Lengend Snippet: Cellular localization of p-mTOR immunoreactivity in the spinal cord dorsal horn. (A-I) Cell-type-specific immunolabeling of p-mTOR in the ipsilateral dorsal horn at 2 h after intraplantar BmK I injection. Arrows indicate colocalization of the p-mTOR (red) with the respective cell markers (green). (J) Histogram of the cellular distribution of p-mTOR.

Article Snippet: The membranes were incubated with the following primary antibodies which were diluted in PBS-T containing 5% nonfat milk for 5 h at room temperature: rabbit polyclonal antibody against p-mTOR (Ser2448, 1:1000; #2971; Cell Signaling Technology, MA [ ])/rabbit monoclonal antibody against mTOR (7C10, 1:1200; #2983; Cell Signaling Technology, MA)/rabbit polyclonal antibody against p-p70 S6K (Thr421/Ser424, 1:1500; sc-7984-R; Santa Cruz, CA [ ])/rabbit monoclonal antibody against p-4E-BP1 (Thr37/46, 1:1000; #2855; Cell Signaling Technology, MA)/rabbit polyclonal antibody against Actin (I-19, 1:1000; sc-1616; Santa Cruz, CA).

Techniques: Immunolabeling, Injection

Journal: Molecular Pain

Article Title: Activation of mammalian target of rapamycin mediates rat pain-related responses induced by BmK I, a sodium channel-specific modulator

doi: 10.1186/1744-8069-9-50

Figure Lengend Snippet: 200 μM rapamycin inhibited the activation of spinal mTOR, 4E-BP1 and p70 S6K assessed by Western blot. (A) & (F) , representative western blots showing levels of p-mTOR, p-4E-BP, p-p70 S6K, total mTOR and β-actin in ipsi- (A) and contralateral (B) side of the spinal cord. (B - E) & (G - J) , histograms represent the mean levels with respect to each saline-treated group at 2 h after i.pl. BmK I injection. The data are presented as mean ± S.E.M. of three rats per group. N.s., ** p <0.01, *** p <0.001, compared with saline-treated group by One-way ANOVA, followed by Bonferroni's post hoc test.

Article Snippet: The membranes were incubated with the following primary antibodies which were diluted in PBS-T containing 5% nonfat milk for 5 h at room temperature: rabbit polyclonal antibody against p-mTOR (Ser2448, 1:1000; #2971; Cell Signaling Technology, MA [ ])/rabbit monoclonal antibody against mTOR (7C10, 1:1200; #2983; Cell Signaling Technology, MA)/rabbit polyclonal antibody against p-p70 S6K (Thr421/Ser424, 1:1500; sc-7984-R; Santa Cruz, CA [ ])/rabbit monoclonal antibody against p-4E-BP1 (Thr37/46, 1:1000; #2855; Cell Signaling Technology, MA)/rabbit polyclonal antibody against Actin (I-19, 1:1000; sc-1616; Santa Cruz, CA).

Techniques: Activation Assay, Western Blot, Saline, Injection

Journal: Nutrients

Article Title: Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response

doi: 10.3390/nu16010084

Figure Lengend Snippet: Tyrosine enhances leucine-induced S6K phosphorylation in C2C12 myoblasts. ( A ) S6K phosphorylation (Thr389) after 15 min of stimulation with 2.5 mM Leu and 2.5 mM of each amino acid ( n = 9) as detected through the alpha screen assay. ( B ) S6K phosphorylation in response to treatment with Leu and/or Tyr or Phenylalanine (Phe) ( n = 3) as detected through the alpha screen assay. ( C ) Dose–response evaluation of S6K phosphorylation at Leu and Tyr concentrations ranging from 0 to 10 mM and from 0 to 2.5 mM ( n = 3), respectively, as detected through the alpha screen assay. ( D ) Representative images of anabolic signaling pathways detected in myoblasts on day 1 post-differentiation through western blotting following 15 min of stimulation with Leu and/or Tyr. Phosphorylation ratios for S6K (Thr 389) ( E ), 4E-BP (Thr37/46) ( F ), mTOR (Ser2448) ( G ), and S6 (Ser235/236, Ser 240/244) ( H , I ) were calculated by dividing the phosphorylation levels by the protein expression levels ( n = 4). All data represent the fold change with respect to the control (0 mM Leu and 0 mM Tyr). Data are presented as mean ± SEM. Circles represent individual values. * p < 0.05, ** p < 0.01 vs. control † p < 0.05, ‡ p < 0.01 vs. 1 mM Leu. § p < 0.05, || p < 0.01 vs. 5 mM Leu as determined by one-way ANOVA ( A , E – I ). Different letters (a–f) represent significant differences ( p < 0.05) as determined by one-way ANOVA ( B ). ** p < 0.01 vs. the Leu alone condition as determined by two-way ANOVA ( C ).

Article Snippet: Subsequently, the membranes were incubated overnight with anti-S6K (9202; Cell Signaling Technology (CST), Danvers, MA, USA), anti-P-S6K (Thr389, 9205; CST), anti-4E-BP (9452; CST), anti-P-4E-BP (Thr37/46, 2855; CST), anti-mTOR (2972; CST), anti-P-mTOR (Ser2448, 2971; CST), anti-P-S6 (Ser235/236, 2211; Ser240/244, 2215; CST), anti-S6 (2217; CST), anti-GAPDH (2118, CST), anti-LRS (13868, CST), anti-Sestrin1 (21668-1-AP, Proteintech, Rosemont, IL, USA), and anti-Sestrin2 (10795-1-AP, Proteintech) antibodies, diluted at a 1:2000 ratio using an immunoreaction-enhancing solution (Can Get Signal, TOYOBO).

Techniques: Western Blot, Expressing

Journal: Nutrients

Article Title: Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response

doi: 10.3390/nu16010084

Figure Lengend Snippet: Effect of leucine and tyrosine on S6K and 4E-BP phosphorylation in isolated muscles. Overnight-fasted male C57BL/6J mice were sacrificed, and their soleus and extensor digitorum longus (EDL) muscles were isolated and incubated in Krebs Ringer Bicarbonate (KRB) buffer containing 0–3 mM Leu and/or 0–1 mM Tyr for 20 min. ( A , B ) S6K phosphorylation (Thr389) levels in the soleus and EDL muscles. ( C , D ) 4E-BP phosphorylation (Thr37/46) levels in the soleus and EDL muscles. All data represent the fold change with respect to the control (0 mM Leu and 0 mM Tyr). Data are presented as mean ± SEM ( n = 8–9). Circles represent individual values. The number of samples in each group is indicated under the bar charts. * p < 0.05, ** p < 0.01 vs. control. † p < 0.05, ‡ p < 0.01 vs. 1 mM Leu as determined by one-way ANOVA.

Article Snippet: Subsequently, the membranes were incubated overnight with anti-S6K (9202; Cell Signaling Technology (CST), Danvers, MA, USA), anti-P-S6K (Thr389, 9205; CST), anti-4E-BP (9452; CST), anti-P-4E-BP (Thr37/46, 2855; CST), anti-mTOR (2972; CST), anti-P-mTOR (Ser2448, 2971; CST), anti-P-S6 (Ser235/236, 2211; Ser240/244, 2215; CST), anti-S6 (2217; CST), anti-GAPDH (2118, CST), anti-LRS (13868, CST), anti-Sestrin1 (21668-1-AP, Proteintech, Rosemont, IL, USA), and anti-Sestrin2 (10795-1-AP, Proteintech) antibodies, diluted at a 1:2000 ratio using an immunoreaction-enhancing solution (Can Get Signal, TOYOBO).

Techniques: Isolation, Muscles, Incubation