Journal: Molecular medicine reports

Article Title: Resveratrol improves neurological outcome and neuroinflammation following spinal cord injury through enhancing autophagy involving the AMPK/mTOR pathway.

doi: 10.3892/mmr.2018.9194

Figure Lengend Snippet: Figure 7. Resveratrol activates autophagy via the AMPK/mTOR signaling pathway. (A) Western blot bands and quantitative analysis of protein levels of AMPK, p‑AMPK, mTOR and p‑mTOR at 3 days post‑SCI. (B) Western blot bands and quantitative analysis of proteins levels of AMPK, p‑AMPK, mTOR, and p‑mTOR at 21 days post‑SCI. β‑actin was used as the loading control and for band density normalization. Values are presented as the mean ± standard devia tion. *P<0.05, vs. sham group; #P<0.05, vs. SCI group. SCI, spinal cord injury; Res, resveratrol; AMPK, AMP‑activated protein kinase; mTOR, mammalian target of rapamycin; p‑, phosphorylated.

Article Snippet: Following blocking with 5% non‐fat milk for 2 h at 37 ̊C, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit anti-LC3 antibody, and rabbit anti-Beclin-1 antibody (cat. no. 3738), rabbit anti-AMPK antibody (cat. no. 5831), rabbit anti-p-AMPK antibody (cat. no. 2535), rabbit anti-mTOR antibody (cat. no. 2983) rabbit anti-p-mTOR antibody (cat. no. 2971) and rabbit anti-β-actin (cat. no. 8457) from Cell Signaling Technology, Inc. (Danvers, MA, USA) at 1:1,000 dilutions.

Techniques: Western Blot, Control

Journal: Molecular medicine reports

Article Title: Resveratrol improves neurological outcome and neuroinflammation following spinal cord injury through enhancing autophagy involving the AMPK/mTOR pathway.

doi: 10.3892/mmr.2018.9194

Figure Lengend Snippet: Figure 8. 3‑MA treatment regulates the AMPK/mTOR signaling pathway. Western blot bands and quantitative analysis of p‑AMPK and p‑mTOR at 3 days post‑SCI. β‑actin was used as the loading control and for band density normalization. Values are presented as the mean ± standard deviation. *P<0.05 vs. sham group; #P<0.05 vs. SCI group. SCI, spinal cord injury; Res, resveratrol; 3‑MA, 3‑methyladenine; AMPK, AMP‑activated protein kinase; mTOR, mammalian target of rapamycin; p‑, phosphorylated

Article Snippet: Following blocking with 5% non‐fat milk for 2 h at 37 ̊C, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit anti-LC3 antibody, and rabbit anti-Beclin-1 antibody (cat. no. 3738), rabbit anti-AMPK antibody (cat. no. 5831), rabbit anti-p-AMPK antibody (cat. no. 2535), rabbit anti-mTOR antibody (cat. no. 2983) rabbit anti-p-mTOR antibody (cat. no. 2971) and rabbit anti-β-actin (cat. no. 8457) from Cell Signaling Technology, Inc. (Danvers, MA, USA) at 1:1,000 dilutions.

Techniques: Western Blot, Control, Standard Deviation

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: AKT/mTOR dependent signaling is involved in the enhancement of cardiac autophagy by DGKζ deficiency Cardiomyocytes infected with lentivirus expressing DGKζ shRNA were subjected to ET-1 for 24h. The total protein expression and phosphorylated level of AKT, mTOR, AMPK, ULK1 and Beclin1 were examined by western blotting, respectively. (A) Representative blots depicting total and phosphorylated proteins. (B) The analyzed results of p-AKT/AKT level, p-mTOR/mTOR level, p-AMPK/AMPK level, p-ULK1/ULK1 level and p-Beclin1 level (one-way ANOVA, n =5, *P< 0.05 vs. control. # P< 0.05 or ## P< 0.01 vs. ET).

Article Snippet: The membranes were blocked and detected with anti-LC3 antibody (Cat#3868), anti-Beclin-1 antibody (Cat#3738), anti-phosphorylated-Beclin1 antibody (Cat#13825), anti-p62 antibody (Cat#5114), anti-phosphorylated-Akt antibody (Cat#9271), anti-Akt antibody (Cat#9272), anti-phosphorylated-AMPK antibody (Cat#2531), anti-AMPK antibody (Cat#2532), anti-phosphorylated-mTOR antibody (Cat#2971), anti-mTOR antibody (Cat#2972), Phospho-ULK1Ser757 antibody (#14202), ULK1 antibody (#8054) from Cell Signaling Technology (Beverly, MA), anti-DGKζ antibody (Cat#SC-8722, Santa Cruz, CA, USA) or anti-β-actin antibody (Cat#BS6007M, Bioworld Technology, St. Louis Park, MN, USA), respectively.

Techniques: Infection, Expressing, shRNA, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Deficiency of DGKζ impaired the interaction of mTOR with TFEB and favored the nuclear translocation of TFEB. (A) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in heart tissues after TAC (Student t test, n =7-8, * P < 0.05 or ** P < 0.01 vs. sham. The expression of proteins in TAC mice was normalized to that of the sham mice in each time point, and β-actin served as a loading control). (B) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in ET-1-treated cardiomyocytes (one-way ANOVA, n =6, * P <0.05 vs. 0h). (C) Immunofluorescent staining showing the translocation of TFEB from cytoplasm to nucleus induced by ET-1. (D) The representative image of immunoprecipitation assay showing that mTOR interacted with TFEB in cardiomyocytes. Input was used as a positive control and IgG was used as a negative control. (E) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in cardiomyocytes after DGKζ knockdown (one-way ANOVA, n =5-6, * P <0.05 vs. control, # P< 0.05 vs. ET). (F) Immunofluorescent staining showing the translocation of TFEB from cytoplasm to nucleus induced by ET-1 after DGKζ knockdown.

Article Snippet: The membranes were blocked and detected with anti-LC3 antibody (Cat#3868), anti-Beclin-1 antibody (Cat#3738), anti-phosphorylated-Beclin1 antibody (Cat#13825), anti-p62 antibody (Cat#5114), anti-phosphorylated-Akt antibody (Cat#9271), anti-Akt antibody (Cat#9272), anti-phosphorylated-AMPK antibody (Cat#2531), anti-AMPK antibody (Cat#2532), anti-phosphorylated-mTOR antibody (Cat#2971), anti-mTOR antibody (Cat#2972), Phospho-ULK1Ser757 antibody (#14202), ULK1 antibody (#8054) from Cell Signaling Technology (Beverly, MA), anti-DGKζ antibody (Cat#SC-8722, Santa Cruz, CA, USA) or anti-β-actin antibody (Cat#BS6007M, Bioworld Technology, St. Louis Park, MN, USA), respectively.

Techniques: Translocation Assay, Expressing, Control, Staining, Immunoprecipitation, Positive Control, Negative Control, Knockdown

Journal:

Article Title: Aerobic Exercise Overcomes the Age-Related Insulin Resistance of Muscle Protein Metabolism by Improving Endothelial Function and Akt/Mammalian Target of Rapamycin Signaling

doi: 10.2337/db06-1566

Figure Lengend Snippet: Phosphorylation of Akt/PKB, mTOR, 4E-BP1, and S6K1 in two groups of older subjects at baseline and during insulin infusion performed ~20 h following rest (Control: n = 5 for Akt/PKB and mTOR; n = 4 for 4E-BP1 and S6K1) or aerobic exercise (Exercise: n = 6). We used the following phospho and total primary antibodies (Cell Signaling, Beverly, MA): phospho-mTOR (Ser2448, cat. no. 2971, lot no. 9; 1:1,000), phospho-p70 S6K1 (Thr389, cat. no. 9234, lot no. 2; 1:500), phospho-Akt (Ser473, cat. no. 4058, lot no. 6; 1:500), and phospho-4EBP1 (Thr37/46, cat. no. 2971; 1:1,000). Anti-rabbit IgG horseradish peroxidase–conjugated secondary antibody was purchased from Amersham Bioscience (1:2,000). Values are means ± SE. *P < 0.05 vs. basal, #P < 0.05 vs. control. □, basal; ■, insulin. Bas, basal; Ins, insulin; MW, molecular weight.

Article Snippet: We used the following phospho and total primary antibodies (Cell Signaling, Beverly, MA): phospho-mTOR (Ser 2448 , cat. no. 2971, lot no. 9; 1:1,000), phospho-p70 S6K1 (Thr 389 , cat. no. 9234, lot no. 2; 1:500), phospho-Akt (Ser 473 , cat. no. 4058, lot no. 6; 1:500), and phospho-4EBP1 (Thr 37/46 , cat. no. 2971; 1:1,000).

Techniques: Phospho-proteomics, Control, Molecular Weight

Journal: Molecular Medicine Reports

Article Title: Platelet-derived growth factor D promotes the angiogenic capacity of endothelial progenitor cells

doi: 10.3892/mmr.2018.9692

Figure Lengend Snippet: PDGF-D functions via PDGFR-dependent and -independent pathways. (A-E) Western blot analysis was performed on the total protein extracted from confluent PDGF-D-EPCs and GFP-EPCs. (1–1, 1–2, 1–3 stand for GFP-EPCs and 2–1, 2–2, 3–3 represent PDGF-D-EPCs). The expression and phosphorylation of STAT3, AKT, ERK1/2, mTOR and GSK-3β were measured by immunoblotting, and normalized to the levels of GAPDH. **P<0.01 vs. the control group. (F) The expression of VEGF, HGF and PDGF-B was examined by RT-qPCR. The expression levels were normalized to GAPDH; **P<0.01 vs. the control group.

Article Snippet: The following antibodies (Abs) (Cell Signaling Technology, Inc., Danvers, MA, USA) were used (all dilutions, 1:1,000): GSK-3β Ab (cat no. 9315), phospho-GSK-3β Ab (cat no. 9323), STAT3 Ab (cat no. 8232), phospho-STAT3 Ab (cat no. 9134), mTOR Ab (cat no. 2972), phospho-mTOR Ab (cat no. 2971), ERK1/2 Ab (cat no. 4696), phospho-ERK1/2 Ab (cat no. 8544) and PDGF-D Ab (SCBT, Dallas, TX, USA).

Techniques: Western Blot, Expressing, Phospho-proteomics, Control, Quantitative RT-PCR

Journal: The Journal of Physiology

Article Title: Simulated altitude is medicine: intermittent exposure to hypobaric hypoxia and cold accelerates injured skeletal muscle recovery

doi: 10.1113/jp285398

Figure Lengend Snippet: Figure 6. Expression of Akt and pSer473Akt proteins in the injured and non-injured gastrocnemius muscles A and B, total Akt; C and D, pSer473Akt; and E and F, pSer473Akt to total Akt ratio. Data are presented as mean ± standard deviation and expressed in arbitrary units (a.u.). Points represent the values of each sample; n = 4–5 per group at each time point. Statistically significant differences are indicated as: B), bNon-Injured: HYPO vs. COLD: P = 0.011. C, aInjured: COHY vs. CTRL: P = 0.027; Non-Injured: HYPO vs. CTRL: P = 0.046 and COHY vs. CTRL: P = 0.009. E, aInjured: HYPO vs. CTRL: P = 0.015 and COHY vs. CTRL: P = 0.024; Non-Injured: COHY vs. CTRL: P = 0.025. CTRL, control; COLD, intermittent cold; HYPO, intermittent hypobaric hypoxia; COHY, simultaneous intermittent cold and hypobaric hypoxia.

Article Snippet: To detect our target proteins, membranes were incubated overnight at 4°Cwith primary antibodies against phosphorylated forms pSer2448mTor, pThr172AMPKα and pSer473Akt (Cat. no. 2971, RRID: AB_330 970; Cat. no. 2535, RRID: AB_331 250; Cat. no. 4060, RRID: AB_2 315 049, Cell Signaling Technology, Danvers, MA, USA) diluted 1:500 in 4% BSA-TBS-T.

Techniques: Expressing, Muscles, Standard Deviation, Control

Journal: The Journal of Physiology

Article Title: Simulated altitude is medicine: intermittent exposure to hypobaric hypoxia and cold accelerates injured skeletal muscle recovery

doi: 10.1113/jp285398

Figure Lengend Snippet: Figure 9. Representative images of the expression of Akt, pSer473Akt, AMPKα, pThr172AMPKα, mTOR and pSer2448mTOR proteins in injured (I) and non-injured (N-I) gastrocnemius muscles, and an example of loading control (Ponceau staining) The figure is composed of the combination of different bands coming from different membranes, as indicated by lines, that were selected as representative according to the results of the statistical analysis of our data. CTRL, control; COLD, intermittent COLD; HYPO, intermittent hypobaric hypoxia; COHY, simultaneous intermittent cold and hypobaric hypoxia.

Article Snippet: To detect our target proteins, membranes were incubated overnight at 4°Cwith primary antibodies against phosphorylated forms pSer2448mTor, pThr172AMPKα and pSer473Akt (Cat. no. 2971, RRID: AB_330 970; Cat. no. 2535, RRID: AB_331 250; Cat. no. 4060, RRID: AB_2 315 049, Cell Signaling Technology, Danvers, MA, USA) diluted 1:500 in 4% BSA-TBS-T.

Techniques: Expressing, Muscles, Control, Staining