total foxo1  (Cell Signaling Technology Inc)

Journal: FASEB BioAdvances

Article Title: Effect of Denervation on Skeletal Muscle Mitochondria in Heterozygous mtDNA Mutator Mice

doi: 10.1096/fba.2025-00072

Figure Lengend Snippet: Autophagy and ubiquitin‐proteasome‐related proteins and transcripts in response to denervation in WT and PolG (+/mut) mice. (A) Representative images of western blot analysis. Protein expression of (B) p62, (C) Parkin, (D) BNIP3, (E) LC‐3I, (F) LC‐3II, (G) LC‐3II/I, (H) TNF‐α, (I) phosphorylated/total FoxO1, (J) MuRF1, (K) MAFbx and (L) Ubiquitin‐conjugated proteins. mRNA expression levels of (M) Sqstm1 , (N) Park2 , (O) Murf1 and (P) Mafbx . All data are expressed as mean ± SD with individual values ( n = 6 per group). Paired data are connected with lines. Significant differences were assessed using a two‐way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: The following primary antibodies were used for western blotting: PGC‐1α (1:1000; 516,557; Millipore), cytochrome C (1:1000; 556433; BD Bioscience), oxidative phosphorylation (OXPHOS); NDUFB8 (NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8), SDHB (succinate dehydrogenase complex subunit B), UQCRC2 (ubiquinol‐cytochrome c reductase core protein II), ATP5A (ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1) (1:3000; ab110413; Abcam), mitofusin 2 (MFN2) (1:1000; ab124773; Abcam), optic atrophy protein 1 (OPA1) (1:1000; #612606; BD Biosciences), dynamin‐related protein 1 (DRP1) (1:1000; ab56788; abcam), p‐DRP1 (1:1000; #3445; Cell Signaling Technology), mitochondrial fission protein 1 (FIS1) (1:1000; ab96764; Abcam), tumor necrosis factor alpha (TNF‐α) (1:1000; sc‐130349; Santa Cruz), ubiquitin‐conjugated proteins (1:1000; sc‐166553; Santa Cruz), forkhead box protein O1 (p‐FoxO1) (Ser256, 1:1000; #9461; Cell Signaling Technology), total‐FoxO1 (1:1000; #2880; Cell Signaling Technology), muscle RING‐finger protein (MuRF1) (1:1000; sc‐32920; Santa Cruz), muscle atrophy F‐box (MAFbx) (1:1000; sc‐32920; Santa Cruz), microtubule‐associated protein 1 light chan 3 (LC3) (1:1000; #4108; Cell Signaling Technology), sequestosome 1 (p62/SQSTM1) (1:1000; PM045; MBL), E3 ubiquitin‐protein ligase parkin (Parkin) (1:1000; ab77924; Abcam), Bcl‐2/E1B‐19 kDa‐interacting protein 3 (Bnip3) (1:1000; sc‐56167; Santa Cruz), 4‐hydroxynonenal (4‐HNE) (1:1000; ab46545; Abcam), Heme oxygenase (HO‐1) (1:1000; #70081; Cell Signaling Technology), and Catalase (1:1000; sc‐271803; Santa Cruz).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Comparison

Journal: Kidney Research and Clinical Practice

Article Title: Pan-Nox inhibitor treatment improves renal function in aging murine diabetic kidneys

doi: 10.23876/j.krcp.23.004

Figure Lengend Snippet: (A) Representative immunoblots for plasminogen activator inhibitor 1 (PAI-1) and type IV collagen (Col4); (B) densitometric analysis of immunoblots for PAI-1 and Col4; (C) representative immunoblot for Klotho, p-FoxO1, t-FoxO1 and Nrf2; and (D) densitometric analysis of immunoblot for Klotho, p-FoxO1/t-FoxO1 ratio, Nrf2. Data are shown as the mean ± standard error of mean. * p < 0.05, p < 0.001 vs. control or APX-115, # p < 0.05, ### p < 0.001 vs. streptozotocin (STZ).

Article Snippet: Proteins were transferred onto a polyvinylidene difluoride membrane, and the membrane was hybridized in blocking buffer overnight at 4 °C with rabbit polyclonal anti-Nox1 antibody (1:1,000; Abcam Plc), rabbit polyclonal anti-Nox2 antibody (1:1,000; Bioworld Technology), rabbit polyclonal anti-Nox4 antibody (1:1,000; Bioworld Technology), rabbit polyclonal anti-PAI-1 antibody (1:1,000; Santa Cruz Biotechnology), mouse monoclonal anti-monocyte chemoattractant protein (MCP)-1 antibody (1:1000; Lsbio), rabbit polyclonal anti-type IV collagen antibody (1:1,000; Abcam Plc), rabbit polyclonal anti-TGF-β1 antibody (1:1,000; Abbkine), goat polyclonal anti-Klotho antibody (1:1,000; R&D Systems Inc.), rabbit polyclonal anti-phospho FoxO1 antibody (1:1,000; Lsbio), rabbit polyclonal anti-total-FoxO1 antibody (1:1,000; Cell Signaling Technology), rabbit polyclonal anti-NRF2 antibody (1:1,000; Cell Signaling Technology), or mouse monoclonal anti-β actin antibody (1:5,000; Sigma-Aldrich).

Techniques: Western Blot, Control

Journal: Frontiers in Immunology

Article Title: CARD11 regulates the thymic Treg development in an NF-κB-independent manner

doi: 10.3389/fimmu.2024.1364957

Figure Lengend Snippet: CARD11 regulates tTreg development through the AKT/FOXO1 pathway. (A, B) Naïve CD4 + T cells from WT and E134G mice and (B) WT and K215M mice were stimulated with anti-CD3 and anti-CD28 (2μg/mL) at different time points. The phosphorylations of AKT (T308), AKT (S473), and FOXO1 were measured by western blot. (C) CD4 + SP Thymocytes of WT and E134G mice, as well as (D) WT and K215M mice, were intracellularly stained for p-AKT (T308) and analyzed by flow cytometry (left). The quantifications of pAKT (T308) MFI in WT and Card11 mutant mice (right) (n=3). (E) WT and E134G BM hematopoietic stem cells were transduced with empty retrovirus or FOXO1 overexpression retrovirus, respectively, and then applied to BM chimeras. Six weeks later, GFP + CD4 + CD25 + FoxP3 + Treg cells in the spleens of recipient mice were analyzed by flow cytometry (left). The quantifications of Treg cells are shown in the right panel. (n=5). Data represent the mean ± SEM of n>3 biological replicates. *P<0.05, ***P<0.001; P values were determined using unpaired two-tailed Student’s t- tests.

Article Snippet: After transfer, nitrocellulose membranes were blocked in 5% milk for up to 1 hour at room temperature, followed by incubation with primary antibodies against phosphor-AKT (T308), phosphor-AKT (S473), phosphor-FOXO1, total FOXO1, total AKT, and mouse anti-actin (Cell Signaling Technology) at 4°C overnight.

Techniques: Western Blot, Staining, Flow Cytometry, Mutagenesis, Transduction, Over Expression, Two Tailed Test