Journal: International Journal of Oncology

Article Title: MDIG, a 2-oxoglutarate-dependent oxygenase, acts as an oncogene and predicts the prognosis of multiple types of cancer

doi: 10.3892/ijo.2022.5372

Figure Lengend Snippet: Regulation of the mTORC2/Akt and PDK1/Akt signaling pathway by MDIG. Western blot analysis was used to examine the mTORC2/Akt and PDK1/Akt signaling pathways. GAPDH was used as the loading control. Phosphoproteins were presented as the ratio of phosphoprotein to total protein. * P<0.05, ** P<0.01 vs. Con or Vector group (one-way ANOVA followed by Tukey's post-hoc test). MDIG, mineral dust-induced gene; Con, control; LV, lentiviral vector; shRNA, short hairpin RNA; P, phosphorylated; PDK1, pyruvate dehydrogenase kinase 1; mTORC2, mTOR complex 2.

Article Snippet: The membranes were subsequently washed with 1X Tris-buffered saline containing 0.1% Tween ® -20 detergent (1X TBST) and then incubated with primary antibodies against MINA53 (cat. no. sc-398521), cyclin-dependent kinase (CDK)2 (cat. no. sc-6248), CDK6 (cat. no. sc-7961), CDK inhibitor 1A (CDKN1A; cat. no. sc-6246), CDKN2D (cat. no. sc-1665; all at 1:1,000 dilution; mouse monoclonal; Santa Cruz Biotechnology, Inc.), pan-Akt (cat. no. 4691), pyruvate dehydrogenase kinase 1 (PDK1; cat. no. 13037), P-PDK1 (Ser241; cat. no. 3438), P-Akt (Thr308; cat. no. 13038), mTOR (cat. no. 2983), GβL (cat. no. 3274), Rictor (cat. no. 9476), P-Akt (Ser473; cat. no. 4060) and GAPDH (cat. no. 5174; all at 1:1,000 dilution; rabbit monoclonal; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Western Blot, Protein-Protein interactions, Control, Plasmid Preparation, shRNA

Journal: Pediatric research

Article Title: Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets.

doi: 10.1038/s41390-022-02382-4

Figure Lengend Snippet: Fig. 1 Current models of Akt activation. The activation of Akt is highly complex and is tightly controlled via a multistep process that depends on physiological conditions, the cell type, and types of stimuli. 4EBP1 eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1, Akt1/2/3 Akt/PKB protein kinase B 1/2/3, eEF2 elongation factor 2, eIF2α eukaryotic translation initiation factor 2α, eIF4E eukaryotic translation initiation factor 4E, eIF4G eukaryotic translation initiation factor 4G, FAK focal adhesion kinase, IP6K1 inositol hexakisphosphate kinase 1, IP7 5- diphosphoinositolpentakisphosphate, IR insulin receptor, IRS1 insulin receptor substrate 1, mTORC1/2 mechanistic target of rapamycin complex 1/2, PDK1 3-phosphoinositide-dependent protein kinase-1, PI3K phosphatidylinositol-3-kinase, PHLPP PH domain leucine-rich repeat protein phosphatase, PP2A protein phosphatase 2A, PTEN phosphatase and tensin homolog deleted on chromosome ten, rpS6 ribosomal protein S6, S6K1 p70 ribosomal protein S6 kinase 1, SHIP2 SH2 domain-containing inositol phosphatase, TSC1/2, Ulb4A, ubiquitin-like protein 4A.

Article Snippet: The primary antibodies that were used were: phospho-pan-/Akt (Ser472/473/474) (1:1000; #ab192623; Abcam, Cambridge, MA); pan-Akt, Akt1, Akt2, Akt3 (Rabbit mAb #4685, Rabbit mAb #75692, Rabbit mAb #3063, and Mouse mAb #8018), FAK (total, #3285 and Tyr397, #8556), mTOR (total, #2972), PTEN (total, #9552 and Ser308, #9551), PP2A (total, #2260) and PDK1 (total, #3062 and Ser241, #3438) (1:1000; Cell Signaling Technology); PP2A (Tyr307, # AF3989-SP; 1:1000; R&D System, Minneapolis, MN); Vinculin (#66305-1-lg; 1:4000); PHLPP (#676640-1-lg), Ubl4A (#14253-1-AP), IP6K1 (#12057-2-AP), Hsp90 (#13171-1-AP) and GAPDH (#60004-1-lg) (1:1000; Proteintech, Rosemont, IL); phospho-mTOR (Ser2481, #651701) (1:1000; BioLegend).

Techniques: Activation Assay, Binding Assay, Ubiquitin Proteomics

Journal: Pediatric research

Article Title: Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets.

doi: 10.1038/s41390-022-02382-4

Figure Lengend Snippet: Fig. 3 Preterm birth blunts positive regulators of Akt activation. a Western analysis of phosphorylated PDK1 at Ser241. The phosphorylation of mTORC2, Akt-GAPDH complex abundance, and Akt-Hsp90 complex abundance were analyzed by immunoprecipitation followed by Western blot. b–f Western blot analysis of phosphorylated mTORC2 at Ser2481, phosphorylated FAK at Tyr397, Ubl4A abundance, Akt-GAPDH complex abundance, and Akt-Hsp90 complex abundance. Values were corrected by their protein abundance in the immunoprecipitates or tissue lysate samples. Ubl4A abundance values were normalized by GAPDH in the samples. For PDK1, there was a significant interaction (P < 0.001) between GAB and STATE. Representative blots are shown. Data are expressed as mean ± SEM; n = 21–25/ group (*P < 0.05 and **P < 0.01). AU arbitrary units.

Article Snippet: The primary antibodies that were used were: phospho-pan-/Akt (Ser472/473/474) (1:1000; #ab192623; Abcam, Cambridge, MA); pan-Akt, Akt1, Akt2, Akt3 (Rabbit mAb #4685, Rabbit mAb #75692, Rabbit mAb #3063, and Mouse mAb #8018), FAK (total, #3285 and Tyr397, #8556), mTOR (total, #2972), PTEN (total, #9552 and Ser308, #9551), PP2A (total, #2260) and PDK1 (total, #3062 and Ser241, #3438) (1:1000; Cell Signaling Technology); PP2A (Tyr307, # AF3989-SP; 1:1000; R&D System, Minneapolis, MN); Vinculin (#66305-1-lg; 1:4000); PHLPP (#676640-1-lg), Ubl4A (#14253-1-AP), IP6K1 (#12057-2-AP), Hsp90 (#13171-1-AP) and GAPDH (#60004-1-lg) (1:1000; Proteintech, Rosemont, IL); phospho-mTOR (Ser2481, #651701) (1:1000; BioLegend).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Quantitative Proteomics

Journal:

Article Title: Caveolin-1 Maintains Activated Akt in Prostate Cancer Cells through Scaffolding Domain Binding Site Interactions with and Inhibition of Serine/Threonine Protein Phosphatases PP1 and PP2A

doi: 10.1128/MCB.23.24.9389-9404.2003

Figure Lengend Snippet: The expression of cav-1 leads to increased phosphorylation of multiple Akt substrates. The phosphorylation state of Akt substrates was analyzed using Tg-treated cav-1-expressing and control cells. Data used were from two independent viral infection experiments (indicated as #1 and #2). The numbers under each gel band are the ratios of phosphorylated protein (indicated by P- prefix before the protein name) to β-actin in the same sample. The protein expression of corresponding molecules in the same cell lysates are also shown. Note significantly increased phosphorylation levels of GSK3α/β, FKHR, and MDM2 in cav-1-expressing cells. Marginally increased phosphorylation of BAD and IKKα/β were also observed.

Article Snippet: Rabbit polyclonal antibodies against IKKα, IKKβ, phospho-Akt (Ser473), phospho-Akt (Thr308), phospho-IKKα (Ser180)/IKKβ (Ser181), phospho-MDM2 (Ser166), phospho-FKHR (Thr24)/FKHRL1 (Thr32), phospho-tyrosine (P-Tyr-100), monoclonal antibody against phospho-CREB (Ser133), and Akt kinase assay kit were from Cell Signaling.

Techniques: Expressing, Infection