Journal: Nature communications

Article Title: Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis.

doi: 10.1038/ncomms9137

Figure Lengend Snippet: Figure 2 | UCP3 overexpression inhibits Akt activation. (a) Immunoblot for Akt phosphorylation at Ser 473, along with total Akt, Cyclin D1 and Cyclin A expression, and cell cycle inhibitory proteins p21 (Cip1/Waf1) and p27 (Kip1) expression following treatment with acetone (vehicle control, 4-h post treatment) or 2.5 mg TPA (4 and 18-h post treatment) in wild-type FVB and K5-UCP3 epidermal lysates. (b) Akt phosphorylation at Ser 473 and Thr 308, along with phosphorylation of Akt targets FOXO1 at Ser 256 and Ser 319, and GSK3b at Ser 9 in wild-type FVB and K5-UCP3 epidermal lysates, following topical treatment with 2.5 mg TPA or acetone. (c) Phosphorylation of mTORC1 pathway members TSC2 (Ser 939), mTOR (Ser 2448, Ser 2481), 4EBP1 (Thr 36/47), ribosomal S6 (Thr 240/244) and eIF4G (Ser 1108) in wild-type FVB and K5-UCP3 epidermal lysates following topical treatment with 2.5-mg TPA or acetone. (d) Phosphorylation of Akt Ser 473 and (e) phosphorylation of EGFR (Ser 1086) in serum starved wild-type FVB and K5-UCP3 primary epidermal keratinocytes, after treatment with 40 ng ml 1 EGF or 0.001% BSA (vehicle control) at the indicated time points. (f) Phosphorylation of Akt Ser 473 in primary neonatal human keratinocytes transiently transfected with UCP3 or empty vector control (EV) and treated with 40 ng ml 1 EGF or 0.001% BSA. Immunoblot for UCP3 confirmed successful transfection. (g) In vitro PP2A catalytic activity, expressed as fold change compared with wild type. Immunoprecipitated PP2Ac was incubated with a target phosphopeptide and free phosphate release was measured using a malachite green assay and absorbance at 620 nm. Error bars represent means þ / s.e.m. (n ¼ 4 biological replicates). ***Indicates significantly different from wild type (Po0.001, Student’s t-test). (h) Immunoblot for Akt Ser 473 and p38 MAPK T180/Y182 phosphorylation in wild-type FVB and K5-UCP3 epidermal lysates 1 h following topical treatment with 5-nmol okadaic acid or acetone (vehicle control). b-Actin or a-Tubulin confirmed equal loading (a-f,h).

Article Snippet: Blots were probed with the following primary antibodies: a-phospho Akt S473 (Cell Signaling Technology (CST) 9271S, Rabbit, 1:1,000), a-phospho Akt T308 (CST 9275S, Rabbit, 1:1,000), a-Akt (CST 9272S, Rabbit, 1:1,000), a-phospho FOXO1 S256 (CST 9461, Rabbit, 1:1,000), a-phospho FOXO1 S319 (CST 2486, Rabbit, 1:1,000), a-phospho GSK-3b S9 (CST 5558, Rabbit, 1:1,000), a-GSK-3b (CST 12456, Rabbit, 1:1,000), a-phospho TSC2 S939 (CST 3615, Rabbit, 1:1,000), a-phospho mTOR S2448 (CST 5536, Rabbit, 1:1,000), a-phospho mTOR S2481 (CST 2974, Rabbit, 1:1,000), a-mTOR (CST 2983, Rabbit, 1:1,000), a-phospho 4EBP1 T36/47 (CST 2855, Rabbit, 1:1,000), a-4EBP1( CST 9644P, Rabbit, 1:1,000), a-phospho rs6 T240/244 (CST 5364.

Techniques: Over Expression, Activation Assay, Western Blot, Phospho-proteomics, Expressing, Control, Transfection, Plasmid Preparation, In Vitro, Activity Assay, Immunoprecipitation, Incubation, Malachite Green Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: The role of the chemokine receptor XCR1 in breast cancer cells

doi: 10.2147/BCTT.S126184

Figure Lengend Snippet: Activation of MAPK and PI3K/AKT/mTOR pathways is involved in XCR1-mediated tumor cell. Notes: ( A , C ) Western blot analysis of MAPK and PI3K/AKT/mTOR pathways protein expression in 231/vector and 231/XCR1 cells. ( B , D ) Phospo-P53 and LC3 protein expression were detected by immunohistochemistry analysis in xenografts of 231/vector and 231/XCR1 tumor bearing mice. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase.

Article Snippet: Antibodies against MEK1/2 (9126, 1:1000), Phospho-MEK1/2 (2338, 1:1000), Phospho-ERK1/2 (4376, 1:1000), AKT (4691.1:1000), Phospho-AKT (4060P,1:1000), Phospho-P38 (4511.1:1000), Phospho-JNK (4668,1:1000), E-cadherin (3195,1:1000), Phospho-p53 (9284.1:1000), and mTOR Substrates Sampler kit (CST 9862) that conclude Phospho-mTOR, mTOR, Phospho-p70s6389 (Thr389), Phospho-P70389 (Thr371), Phospho-4EBP1 (Thr37/46), and anti-rabbit IgG were from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Expressing, Plasmid Preparation, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotection of Kaji-Ichigoside F1 via the BDNF/Akt/mTOR Signaling Pathways against NMDA-Induced Neurotoxicity

doi: 10.3390/ijms232416150

Figure Lengend Snippet: The immunofluorescence of NR2B, AMPA, BDNF, AKT, mTOR, PSD95, and Syn1 in PC12 cells. ( A ) Fluorescence images. ( B ) The mean fluorescence intensity of NR2B, # p < 0.01 versus vehicle group, * p < 0.01 versus NMDA group; AMPA, ## p < 0.01 versus vehicle group, * p < 0.01 versus NMDA group; BDNF, ## p < 0.01 versus vehicle group, * p < 0.01 versus NMDA group; AKT, # p < 0.01 versus vehicle group, * p < 0.05, ** p < 0.01 versus NMDA group; mTOR, ## p < 0.01 versus vehicle group, ** p < 0.01 versus NMDA group; PSD95, ## p < 0.01 versus vehicle group, * p < 0.05, ** p < 0.01 versus NMDA group; and Syn1 ## p < 0.01 versus vehicle group, ** p < 0.01 versus NMDA group;. Green fluorescence represents fluorescent staining, and blue fluorescence represents DAPI-stained nuclei. Bars: 100 μm, the data represent mean ± SEM ( n = 5).

Article Snippet: The mean fluorescence intensities of NR2B (1:100 dilution, Abcam, Cambridge, UK), AMPA (1:100 dilution, CST, USA), BDNF (1:100 dilution, Abcam, USA), AKT (1:100 dilution, CST, USA), mTOR (1:100 dilution, CST, USA), PSD95 (1:100 dilution, Abcam, USA), and Syn1 (1:100 dilution, CST, USA) in the NMDA-induced group decreased significantly compared with in the vehicle group ( p < 0.01).

Techniques: Immunofluorescence, Fluorescence, Staining