Journal: Frontiers in Cellular Neuroscience

Article Title: HDAC1 Silence Promotes Neuroprotective Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Mouse Model of Traumatic Brain Injury via PI3K/AKT Pathway

doi: 10.3389/fncel.2018.00498

Figure Lengend Snippet: HDAC1-silenced MSCs modulated the protein expression of PI3K/Akt pathway in hippocampus. (A) Representative Western blot bands of PTEN, phosphorylated PTEN (p-PTEN), AKT1, phosphorylated AKT1 (p-AKT1), GSK-3β, and phosphorylated GSK-3β (p-GSK-3β) in each group. (B) Quantitative protein levels were normalized with β-actin. Data are presented as mean ± SEM. * p < 0.05 vs. Vehicle, # p < 0.05 vs. MSCs.

Article Snippet: The membrane was blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies respectively directed against Bcl2 (1:500, Proteintech, China), Caspase3 (1:500, Proteintech, China), Cleaved Caspase3 (1:500, Proteintech, China), HDAC1 (1:500; Abcam, Cambridge, England), PTEN (1:500; Sangon Biotech, Shanghai, China), Phospho-PTEN (Ser380/Thr382/Thr383; 1:500; Sangon Biotech, Shanghai, China), AKT-1 (1:500; Sangon Biotech, Shanghai, China), Phospho-AKT1 (Ser473; 1:500; Sangon Biotech, Shanghai, China), GSK-3β (1:500; Proteintech, Wuhan, China), Phospho-GSK-3β (Ser9; 1:500; Cell Signaling Technology, American), or β-actin (1:5,000; Sangon Biotech, Shanghai, China) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: HDAC1 Silence Promotes Neuroprotective Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Mouse Model of Traumatic Brain Injury via PI3K/AKT Pathway

doi: 10.3389/fncel.2018.00498

Figure Lengend Snippet: LY294002 abolished the effects of HDAC1-silenced MSCs on neurologic function and the PI3K/AKT pathway of TBI mice. (A) Quantification of lesion volume. (B) Representative immunofluorescent images. (C) Quantification analysis of fluorescence intensity indicated that DCX + (red) cells in the dentate gyrus of the hippocampus in each field, Scale bar = 200 μm. (D) Representative Western blot and (E) densitometry measurement of AKT1, p-AKT1, GSK-3β, and p-GSK-3β in each group at 3 days post-TBI, (F) TST, (G) FST, (H) SPT. Data are presented as mean ± SEM. * p < 0.05 vs. Vehicle, # p < 0.05 vs. MSCs-siHDAC1.

Article Snippet: The membrane was blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies respectively directed against Bcl2 (1:500, Proteintech, China), Caspase3 (1:500, Proteintech, China), Cleaved Caspase3 (1:500, Proteintech, China), HDAC1 (1:500; Abcam, Cambridge, England), PTEN (1:500; Sangon Biotech, Shanghai, China), Phospho-PTEN (Ser380/Thr382/Thr383; 1:500; Sangon Biotech, Shanghai, China), AKT-1 (1:500; Sangon Biotech, Shanghai, China), Phospho-AKT1 (Ser473; 1:500; Sangon Biotech, Shanghai, China), GSK-3β (1:500; Proteintech, Wuhan, China), Phospho-GSK-3β (Ser9; 1:500; Cell Signaling Technology, American), or β-actin (1:5,000; Sangon Biotech, Shanghai, China) at 4°C overnight.

Techniques: Fluorescence, Western Blot

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. 2 Preterm birth attenuates the phosphorylation of Akt1 and Akt2, but not Akt3. The phosphorylation of individual Akt isoform was analyzed by immunoprecipitation followed by western blot. a–c Phosphorylated Akt1 at Ser472, Akt2 at Ser473, and Akt3 at Ser474. Phosphorylation values were corrected by their protein abundance in the immunoprecipitates. For Akt1 and Akt2, there was a significant interaction (P < 0.01) 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: Phospho-proteomics, Immunoprecipitation, Western Blot, Quantitative Proteomics

Journal: Transplantation

Article Title: Protection From Apoptotic Cell Death During Cold Storage Followed by Rewarming in 13-Lined Ground Squirrel Tubular Cells

doi: 10.1097/tp.0000000000000937

Figure Lengend Snippet: FIGURE 3. Protection of squirrel RTECs from apoptosis during CS/REW is associated with increased protein expression of XIAP, pAkt (Ser473), and pBAD (Ser136). A, Representative blots demonstrate increased pAkt (Ser473) and pBAD (Ser136) protein expression in squirrel RTECs subjected to CS/REW versus control squirrel RTECs. The XIAP protein expression persists in squirrel RTECs subjected to CS/REW. In contrast, mouse RTECs subjected to CS/REW have reduced protein expression of the pAkt (Ser473), pBAD (Ser136), and XIAP versus control mouse RTECs. B, The bar graphs represent densitometry values obtained from 4 independent experiments (see methods). Data are shown as means ± SE. No significant difference was found in Akt1 protein expression between squirrel and mouse RTECs. C, pAkt (Ser473) expression was significantly increased in squirrel RTECs subjected to CS/REW compared to control squirrel RTECs and mouse RTECs subjected to CS/REW (*P < 0.001 vs control squirrel RTECs and mouse RTECs subjected to CS/REW). D, pBAD (Ser136) expression was significantly increased in squirrel RTECs subjected to CS/REW compared to control squirrel RTECs and mouse RTECs subjected to CS/REW (*P < 0.001 vs control mouse RTECs and mouse RTECs subjected to CS/REW; #P < 0.05 vs control squirrel RTECs). E, XIAP expression remains persistent in squirrel RTECs subjected to CS/REW compared to control squirrel RTECs and mouse RTECs subjected to CS/REW (*P < 0.001 vs mouse RTECs subjected to CS/REW).

Article Snippet: Equal amounts of protein were separated on SDS-PAGE, transferred onto a nitrocellulose membrane, blocked, and incubated with primary antibodies of cleaved caspase-3 (Cell Signaling, 9661), XIAP (Cell Signaling, 2042), Akt1 (Cell Signaling, 2938) pAkt Ser473 (Cell Signaling, 9271), or pBAD Ser136 (Cell Signaling, 4366) overnight at 4°C.

Techniques: Expressing, Control

Journal: Transplantation

Article Title: Protection From Apoptotic Cell Death During Cold Storage Followed by Rewarming in 13-Lined Ground Squirrel Tubular Cells

doi: 10.1097/tp.0000000000000937

Figure Lengend Snippet: FIGURE 4. Squirrel RTECs treated with Akt1 shRNA and then subjected CS/REW undergo apoptosis. A, Representative blots demonstrate reduced protein expression of Akt1 in squirrel RTECs treated with shRNA against Akt1. Squirrel RTECs deficient in Akt1 and exposed to CS/REW have decreased pAkt (Ser473), pBAD (Ser136) and increased cleaved caspase-3 protein expression compared to wild type control and wild type squirrel RTECs exposed to CS/REW. B, The bar graphs represent densitometry values obtained from 4 indepen- dent experiments (see methods). Data are shown as means ± SE. Akt1 expression was significantly reduced in squirrel RTECs treated with Akt1 shRNA compared to wild type squirrel RTECs (*P < 0.001 vs wild type squirrel RTECs). C, pAkt (Ser473) expression was sig- nificantly reduced in squirrel RTECs treated with Akt1 shRNA compared to wild type squirrel RTECs (*P < 0.05 vs wild type control squir- rel RTECs; *P < 0.001 vs wild type squirrel RTECs subjected to CS/REW; #P < 0.01 vs wild type squirrel control RTECs). D, pBAD (Ser136) expression was significantly reduced in squirrel RTECs treated with Akt1 shRNA compared to wild type squirrel RTECs (*P < 0.01 vs wild type control squirrel RTECs and wild type squirrel RTECs subjected to CS/REW; #P < 0.001 versus wild type control squirrel RTECs). E, Cleaved caspase-3 expression was significantly increased in squirrel RTECs subjected to CS/REW and treated with Akt1 shRNA compared to wild type squirrel RTECs (*P < 0.001 vs wild type and Akt1 deficient control squirrel RTECs and wild type squir- rel RTECs subjected to CS/REW). F, Representative pictures of TUNEL staining in wild type and Akt1 deficient squirrel RTECs exposed to CS/REW is shown. G, Squirrel RTECs deficient in Akt1 and exposed to CS/REW have significantly increased apoptotic cells compared to controls and wild type squirrel RTECs exposed to CS/REW. (*P < 0.001 vs wild type and Akt1 deficient control squirrel RTECs, and wild type squirrel RTECs subjected to CS/REW, n = 4).

Article Snippet: Equal amounts of protein were separated on SDS-PAGE, transferred onto a nitrocellulose membrane, blocked, and incubated with primary antibodies of cleaved caspase-3 (Cell Signaling, 9661), XIAP (Cell Signaling, 2042), Akt1 (Cell Signaling, 2938) pAkt Ser473 (Cell Signaling, 9271), or pBAD Ser136 (Cell Signaling, 4366) overnight at 4°C.

Techniques: shRNA, Expressing, Control, TUNEL Assay, Staining