Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 6 PSAT1 regulated Akt/GSK3β/β-catenin signaling pathway in PDLSCs. A The phosphorylation level of Erk, Akt, and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs with PSAT1 overexpressed. B The phosphorylation level of Erk, Akt, and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs with PSAT1 knocked down. C Western Blot analysis of β-Catenin protein in nucleus and cytoplasm after PSAT1 was overexpressed. D Western Blot analysis of β-Catenin protein in nucleus and cytoplasm after PSAT1 was knocked down. OEPSAT1: PDLSCs with PSAT1 overexpressed. OENC: control PDLSCs for OEPSAT1 PDLSCs. shPSAT1: PDLSCs with PSAT1 knocked down. shNC: control PDLSCs for shPSAT1 PDLSCs. *p < 0.05; **p < 0.01

Article Snippet: When necessary, Ly294002 (#9901, CST) (an inhibitor of Akt phosphorylation) or SC79 (HY18749, MCE, USA) (a promoter of Akt phosphorylation) was added to the osteogenic medium at concentration of 10 μmol/L or 5 μg/mL, respectively.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 7 LY294002 reversed the effects of overexpressing PSAT1 on the osteogenic differentiation of PDLSCs. A The phosphorylation level of Akt and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs. B The quantitative analysis of ALP activity in PDLSCs after osteogenic induction. C ALP staining of PDLSCs after osteogenic induction for 7 days. D Alizarin red staining of PDLSCs after osteogenic induction for 21 days. E Quantitative analysis of mineralized matrix after osteogenic induction for 21 days. F The protein levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. G The mRNA levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. OEPSAT1: PDLSCs with PSAT1 overexpressed. OENC: control PDLSCs. *p < 0.05; **p < 0.01

Article Snippet: When necessary, Ly294002 (#9901, CST) (an inhibitor of Akt phosphorylation) or SC79 (HY18749, MCE, USA) (a promoter of Akt phosphorylation) was added to the osteogenic medium at concentration of 10 μmol/L or 5 μg/mL, respectively.

Techniques: Phospho-proteomics, Activity Assay, Staining, Control

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 8 SC79 reversed the effects of knocking down PSAT1 on the osteogenic differentiation of PDLSCs. A The phosphorylation level of Akt and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs. B The quantitative analysis of ALP activity in PDLSCs after osteogenic induction. C ALP staining of PDLSCs after osteogenic induction for 7 days. D Alizarin red staining of PDLSCs after osteogenic induction for 21 days. E Quantitative analysis of mineralized matrix after osteogenic induction for 21 days. F The protein levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. G The mRNA levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. shPSAT1: PDLSCs with PSAT1 knocked down. shNC: control PDLSCs. *p < 0.05; **p < 0.01

Article Snippet: When necessary, Ly294002 (#9901, CST) (an inhibitor of Akt phosphorylation) or SC79 (HY18749, MCE, USA) (a promoter of Akt phosphorylation) was added to the osteogenic medium at concentration of 10 μmol/L or 5 μg/mL, respectively.

Techniques: Phospho-proteomics, Activity Assay, Staining, Control

Journal: Nature immunology

Article Title: The sphingosine 1-phosphate receptor S1P₂ maintains the homeostasis of germinal center B cells and promotes niche confinement.

doi: 10.1038/ni.2047

Figure Lengend Snippet: Figure 2 Resistance to apoptosis and greater Akt activation in GC B cells deficient in S1P2, Gα12-Gα13 or p115RhoGEF. (a,b) Frequency of GC B cells with activated caspase-3 (Active Csp3+; a) and TUNEL assay of fragmented DNA (b) in chimeras reconstituted with mixtures of S1pr2+/+ or S1pr2−/− (CD45.2+) plus wild-type (CD45.1+) bone marrow (horizontal axes), then immunized with SRBCs and analyzed after 6–8 d. (c) Flow cytometry analysis of Akt phosphorylated at Thr308 (p-Akt(T308)) in follicular and GC B cells from mixed chimeras as in a,b. Right, mean fluorescence intensity (MFI) in various GC B cell populations. (d) Analysis of Akt phosphorylated at Thr308 in wild-type GC B cells from spleen suspensions left untreated (UT) or treated with JTE-013 alone or JTE-013 and wortmannin (JTE-013 + WMN) for 30 min immediately after isolation. (e) Mean fluorescence intensity of Akt phosphorylated at Thr308 in mLN GC B cells from Sphk1f/−Mx1-CreSphk2−/− or Sphk1f/fMx1-CreSphk2−/− (S1P-deficient) mice or mice retaining at least one wild-type allele of Sphk1 (Control (Ctrl)). (f) Flow cytometry analysis of Akt phosphorylated at Thr308 in GC B cells deficient in both Gα12 and Gα13 (G12-G13–DKO) or in p115RhoGEF (Arhgef1−/−), and control cells from littermates (G12-KO or Arhgef1+/+, respectively) and wild-type (CD45.1+) mice (all mixed–bone marrow chimeras). Right, summary of mean fluorescence intensities. (g) Immunoblot analysis of Akt phosphorylated at Ser473 (p-Akt(S473)) in Ramos cells left untreated or treated for 5 min with S1P in the presence or absence of JTE-013. (h) Analysis of Akt phosphorylated at Thr308 in Ramos cells left untreated or treated for 5 min with S1P (10 nm) alone or in the presence of Y27632 (10 µM), JTE-013 (10 µM) or bpV(pic) (500 nM). (i) Phosphorylation of Akt at Thr308 in Ramos cells treated with S1P alone or in the presence of JTE-013 or Y27632, presented relative to results obtained with untreated cells, set as 1 (dashed line). †P ≤ 0.05, *P ≤ 0.01, **P ≤ 0.001 and ***P ≤ 0.0001 (Student’s t-test). Data are from three experiments with eight to nine mice (a,b), six experiments with twelve to thirteen mice (e) or eight experiments with eight mice (i), or are representative of four experiments with seven mice (c) or three mice (d), or two (f) or three (g) experiments.

Article Snippet: For immunoblot analysis, cells were lysed in prewarmed 2× SDS lysis buffer (100 mM Tris, pH 6.8, 4% (wt/vol) SDS, 10% (vol/vol) glycerol, 2% (vol/vol) β-mercaptoethanol and protease inhibitor ‘cocktail’ (Roche)), then were ultracentrifuged, separated by SDS-PAGE and probed with rabbit antibody to Akt phosphorylated at Ser473 (9271; Cell Signaling Technology) and rabbit antibody to actin (A2066; Sigma).

Techniques: Activation Assay, TUNEL Assay, Flow Cytometry, Fluorescence, Isolation, Control, Western Blot, Phospho-proteomics

Journal: iScience

Article Title: Metabolic rewiring and autophagy inhibition correct lysosomal storage disease in mucopolysaccharidosis IIIB

doi: 10.1016/j.isci.2024.108959

Figure Lengend Snippet:

Article Snippet: mouse anti-phosphorylated Akt monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-514032; RRID: AB_2861344.

Techniques: Recombinant, Modification, Protease Inhibitor, Expressing, Clone Assay, Control, Generated, Knock-Out, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism

doi: 10.1007/s00018-018-2850-8

Figure Lengend Snippet: ORP2-KO inhibits Akt phosphorylation and ORP2 interacts with the Cdc37–Hsp90–Akt effector complex. a Western blot analysis of Akt(Ser473) and GSK-3β(Ser9) after 2 h serum starvation followed by an additional 8 min incubation with 100 nM insulin added in the starvation medium. Data are shown for native HuH7 cells, Cas9 control cells, and the ORP2 knockout cell line. b Western blot analysis of Akt(Ser473) and GSK-3β(Ser9) under basal culture conditions or after 8 min incubation with 100 nM insulin added in the basal medium. The ORP2 knockout cell lines are indicated as KO1, KO2, and the Cas9 controls as Cas9. c Quantification of Akt(Ser473) phosphorylation. The data represent four independent western blots of independent cultures similar to that shown in b. d Quantification of the relative GSK-3β(Ser9) phosphorylation from four independent western blots of independent cultures similar to that shown in b. e Western blot quantification of Akt(Ser473) phosphorylation in HUVECs silenced with ORP2-specific shRNA (shORP2) or shRNA control (shCTRL) followed by an epidermal growth factor stimulation [(EGF) 30 ng/ml for 10 min]. The data represent three independent western blots of independent cultures. f HuH7 cells were lysed and subjected to GST-ORP2 pull-down. Cell lysates (L) and pull-downs (PD) were analyzed by western blotting with anti-Cdc37, anti-Hsp90, and anti-Akt antibodies. Plain GST was used as a negative control. g GST-ORP2 pull-down of HuH7 cells treated with 5 µM Withaferin A. Cell lysates (L) and pull-downs (PD) were analyzed by western blotting with anti-Cdc37, anti-Hsp90, and anti-Akt antibodies. h GST-ORP2 pull-down from HuH7 cell lysates treated with lambda protein phosphatase (λ-PP). Cell lysates (L) and pull-downs (PD) were analyzed by western blotting with anti-Akt antibody. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test)

Article Snippet: To study the role of Akt phosphorylation, cell lysates containing 0.1 mg of total protein were treated with 2000 U of Lambda protein Phosphatase (λ-PP, New England BioLabs, Ipswich, MA, USA) in 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, 1 mM MnCl 2 , and protease inhibitor cocktail (Roche Diagnostics) for 1 h at 30 °C, followed by pull-down assay with GST-ORP2 as described above.

Techniques: Western Blot, Incubation, Knock-Out, shRNA, Negative Control

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: Immunohistology using isoform-specific antibodies revealed distinct expression patterns for each AKT isoform in the hippocampus. AKT1 was mainly expressed in the cell body layers, with the greatest levels in stratum pyramidale of CA1. AKT2 was mostly expressed in specific cells in the molecular layer of the dentate gyrus, CA3 and CA1. AKT3 was also mainly expressed in the cell body layers of the hippocampus and showed strong expression in the hilus and CA3. Bottom panels show single Akt knockout (KO) tissue to validate the specificity of the antibodies.

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Expressing, Knock-Out

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: ( a ) MK2206 and AZD5363 blocked AKT activity effectively, shown by reduced phosphorylation levels of the AKT substrate GSK-3β on serine 9 (pGSK-3β S9), but had different effects on phosphorylation levels of AKT itself on serine 473 (pAKT S473). ( b ) MK2206 (MK) significantly reduced pAKT S473 levels normalized to total AKT (totAKT) levels, while AZD5363 (AZD) significantly increased pAKT S473 levels (MK F (3.8) = 30.93, p<0.0001; AZD F (3.8) = 12.30, p = 0.0023). ( c ) MK had a dose-dependent effect on pGSK-3β S9 levels normalized to total GSK-3β (totGSK-3β) levels, with no effect at 10 µM but significantly reduced levels at 30 and 100 µM compared with vehicle treatment (MK F (3,8) =74.56, p<0.001; post hoc comparisons: veh vs. MK10 p=0.998; veh vs. MK30 p=0.002; veh vs. MK100 p<0.001, MK10 vs. MK30 p=0.002, MK30 vs. MK100 p<0.001). AZD also had a dose-dependent effect on pGSK3β-S9 levels, with significant reduction at 10 µM in addition to the 30 and 100 µM doses compared with vehicle (AZD F (3,8) =39.69, p<0.001; post hoc comparisons: veh vs. AZD10 p=0.002; veh vs. AZD30 p<0.001; veh vs. AZD100 p<0.001; AZD10 vs. AZD100 p=0.01). ( d ) MK and AZD treatment significantly reduced phosphorylation of TSC2 at serine 939 (pTSC2 S939) (MK F (3,9) =10.27, p=0.0041; AZD F (3,9) =7.893, p=0.0089). ( e ) Levels of GluA2 normalized to GAPDH was not significantly altered after MK or AZD treatment (MK F (3, 8) =0.279, p=0.838; AZD F (3,9) =0.378, p=0.771). Veh = vehicle, MK10 = 10 µM, MK30 = 30 µM, MK100 = 100 µM; AZD10 = 10 µM, AZD30 = 30 µM, AZD100 = 100 µM, 3–4 mice/group, *p<0.05.

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Activity Assay, Phospho-proteomics

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: ( a ) AKT1 and AKT2 isoforms showed decreased phosphorylation after MK2206 incubation, while AZD5363 resulted in hyperphosphorylation (AKT1: MK2206 F (3, 8) =24.80, p=0.0002, AZD F (3, 8) =37.03, p<0.0001; AKT2: MK2206 F (3, 8) =10.19, p<0.0042, AZD F (3, 8) =34.67, p<0.0001), 3 mice/group. No feedback on PDK1 was observed after incubation with AKT inhibitors (MK2206 F (3, 8) =1.665, p>0.05; AZD5363 F (3, 8) =0.636, p>0.05). ( b ) Validation of specificity of AKT1 and AKT2 phospho-antibodies. ( c ) AKT3 immunoprecipitation specifically pulls down AKT3, leaving AKT1 and AKT2 isoforms in the supernatant. Treatment with 30 µM MK2006 or AZD5363 leads to AKT3 dephosphorylation or hyperphosphorylation.

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Phospho-proteomics, Incubation, Biomarker Discovery, Immunoprecipitation, De-Phosphorylation Assay

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: ( a ) Inhibiting AKT activity in hippocampal slices with AZD5363 (30 µM) prior to four trains of HFS (100 Hz) resulted in significantly impaired L-LTP in hippocampal area CA1 compared with vehicle treatment ( F (1,24) =6.896, p=0.015), n = 13 slices/group, 5 mice/group. ( b ) AKT inhibitor MK2206 (30 µM) also significantly impaired L-LTP compared with vehicle treatment ( F (1,13) =18.639, p<0.001), n = 7–8 slices/group, 4 mice/group. ( c ) Western blot validation of AKT1, AKT2 and AKT3 removal using cortical tissue from Akt isoform-specific KO mice. β-actin, loading control. ( d ) Akt1 KO mice showed significantly impaired L-LTP in hippocampal area CA1 compared with WT mice ( F (1,28) =5.049, p=0.033), n = 15 slices/group, 6 mice/group. ( e ) Akt2 KO mice displayed similar levels of L-LTP to WT mice ( F (1,20) =1.121, p=0.302), n = 10–12 slices/group, 4–5 mice/group. ( f ) Akt3 KO mice showed similar levels of L-LTP to WT mice ( F (1,20) =0.33, p=0.857), n = 8–14 slices/group, 5–7 mice/group. *p<0.05.

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Activity Assay, Western Blot, Biomarker Discovery, Control

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: ( a ) pGSK-3β levels were significantly reduced in the hippocampus of Akt3 KO mice but normal in Akt1 KO mice ( Akt3 KO t (20) = -4.725, p<0.001; Akt1 KO t (14) = 0.104, p=0.918), 4–6 mice/group. ( b–d ) Hippocampal slices from Akt1 KO, Akt2 KO or Akt3 KO mice showed normal LFS-LTD induced by 900 stimuli of 1 Hz compared to WT slices (p>0.05 for all genotypes), n = 12–20 slices/group, 5–7 mice/group. ( e ) Blocking AKT activity in hippocampal slices with AZD5363 (30 µM) prior to 900 stimuli of 1 Hz did not affect LFS-LTD compared to vehicle-treated slices (p>0.05), n = 12–13 slices/group, 6 mice/group. ( f ) AKT activity blocked by MK2206 (30 µM) also resulted in normal LFS-LTD in hippocampal slices compared to vehicle-treated slices (p>0.05), n = 10–12 slices/group, 5 mice/group.

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Blocking Assay, Activity Assay

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: ( a,b ) Blocking AKT activity using AZD5363 or MK2206 resulted in enhanced mGluR-LTD induced with 3,5 RS-DHPG (100 µM) in hippocampal area CA1 (AZD F (1,23) =5.010, p=0.035; MK F (1,40) =7.216, p=0.010), n = 12–20 slices/group, 5–6 mice/group. ( c ) Blocking PI3K using LY294002 resulted in enhanced mGluR-LTD ( F (1,27) =10.809, p=0.003), n = 13–16 slices/group, 7–8 mice/group. ( d–f ) Akt1, Akt2 or Akt3 KO mice show normal mGluR-LTD compared to WT controls (p>0.05), n = 13–20 slices/group, 5–7 mice/group. *p<0.05.

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Blocking Assay, Activity Assay

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were puromycin (mouse monoclonal 12D10), AKT1 (1:1000, Cell Signaling C73H10), phospho-AKT1 S473 (1:1000, Cell Signaling D7F10), AKT2 (1:1000, LSBio, Seattle, WA, LS-C156232), phospho-AKT2 474 (1:1000, Cell Signaling D3H2), AKT3 (1:1000, Cell Signaling E2B6R), AKT3 (1:1000, Cell Signaling L47B1), phospho-AKT S473 (1:1000, Cell Signaling 736E11), pan-AKT (1:2000, Cell Signaling 11E7), phospho-GSK-3β S9 (1:3000, Cell Signaling 5B3), GSK-3β (1:3000, Cell Signaling 27C10), phospho-TSC2 S939 (1:1000, Cell signaling 3615), TSC2 (1:1000, Cell Signaling 3635), phospho-PDK1 S241 (1:3000, Cell Signaling 3061), PDK1 (1:2000, Cell Signaling 3062), phospho-S6 235/236 (1:2000, Cell Signaling 4856), S6 (1:2000, Cell Signaling 2217), phospho-ERK1/2 (1:4000, Cell Signaling 9101), ERK1/2 (1:4000, Cell Signaling 9102), Mouse anti-GluA2 (1:1000, NeuroMAB, Davis, CA), β-actin (1:10000, Abcam, Cambridge, MA), and GAPDH (1:10000, Cell Signaling 14C10).

Techniques: Western Blot, Immunostaining, Immunoprecipitation, Protease Inhibitor, Software, Imaging, Slice Preparation, Incubation

Journal: Journal of Nanobiotechnology

Article Title: S-RBD-modified and miR-486-5p-engineered exosomes derived from mesenchymal stem cells suppress ferroptosis and alleviate radiation-induced lung injury and long-term pulmonary fibrosis

doi: 10.1186/s12951-024-02830-9

Figure Lengend Snippet: MiR-486-5p-engineered MSC exosomes alleviates pulmonary fibrosis via miR-486-5p-SMAD2-pAkt. A SMAD2 protein expression in lungs of RIPF hACE2 CKI/CKI -SftpcCre + mice at 6 M after irradiation via immunofluorescence, with four mice in each group. Representative images were shown. Scale bar = 200 μm. B Fluorescent quantitation of SMAD2 protein expression (red) in RIPF mice treated with different exosomes. Data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01. C mRNA and D-E protein expression of SMAD2 in MLE-12 cells treated with miR-486-5p mimic (miR-486-5p) after adding TGF-β1 (2 ng/mL). MiR-NC was used as control. Data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01. F MiR-486-5p target site in the SMAD2 3' UTR and the sequence alignment of miR-486-5p and the SMAD2 3' UTR. Mutated bases in the psiCHECK-2 construct are bold. G Luciferase activity after co-transfection of miR-486-5p and SMAD2 into HEK293 cells. MiR-NC was used as a control. Renilla luciferase activity was normalized to firefly luciferase activity. Data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01. H-J Western blot analysis of Akt phosphorylation in MLE-12 cells treated with MSC-Exo or miR-486-MSC-Exo. Insulin was used as an inducer of Akt phosphorylation. LY294002 was used as an inhibitor of Akt phosphorylation. Data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: The results showed that miR-486-MSC-Exo significantly increased Akt phosphorylation, which was inhibited by the Akt phosphorylation inhibitor LY294002 (Cell Signaling Technology).

Techniques: Expressing, Irradiation, Immunofluorescence, Quantitation Assay, Control, Sequencing, Construct, Luciferase, Activity Assay, Cotransfection, Western Blot, Phospho-proteomics

Journal: Oncotarget

Article Title: Hoxa5 increases mitochondrial apoptosis by inhibiting Akt/mTORC1/S6K1 pathway in mice white adipocytes

doi: 10.18632/oncotarget.20521

Figure Lengend Snippet: White adipocytes were pretreated with Ad-Hoxa5 or sh-Hoxa5, MK2206 or rapamycin. (A) Relative phosphorylation levels of Akt Ser473 and mTORC1 Ser2448 with or without MK2206 in each group of white adipocytes (n = 3). (B) Relative protein levels of active Caspase-3 and Bcl-2 with or without MK2206 in each group of white adipocytes (n = 3). (C) Relative phosphorylation levels of mTORC1 Ser2448 and S6K1 Thr389 with or without rapamycin in each group of white adipocytes (n = 3). (D) Relative protein levels of active Caspase-3 and Bcl-2 with or without rapamycin in each group of white adipocytes (n = 3). Values are mean ± SEM. * P < 0.05, ** P < 0.01 compared with the control.

Article Snippet: Briefly, white adipocytes were treated with Akt phosphorylation-specific inhibitor MK2206 (10 μM, Selleck, Houston, USA)and mTORC1 inhibitor rapamycin (10 μM, Selleck, Houston, USA) or DMSO (Promega, Madison, USA) for 6 h, and samples were assessed by Western blot.

Techniques: Phospho-proteomics, Control