anti phospho ser 256 foxo1  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2-mediated thermogenic gene expression may occur through the JNK/FOXO1/UCP1 axis (A) Western blotting and quantification of FOXO1 in sWAT from male WT mice housed at room or cold temperature for 3 h ( n = 4 mice per group). (B) Ucp1 expression in sWAT from male WT mice housed at 4 °C for 0, 3, 6, or 12 h ( n = 4 mice). (C) Ucp1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 4 wells per group). (D) Foxo1 expression in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 4 mice). (E) Western blotting and quantification of nuclear FOXO1 (nFOXO1), cytoplasmic FOXO1 (cFOXO1), and total FOXO1 (tFOXO1) in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (F) Western blotting and quantification of phosphorylated JNK (pJNK) and total JNK (tJNK) in sWAT from male WT mice housed at RT or cold for 3 h ( n = 3 mice). (G) Foxo1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 100 μM JNK inhibitor (SP600125) for 6 h ( n = 6 wells per group). (H) Western blotting and quantification of pJNK and tJNK in sWAT from control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (I) Foxo1 and (J) Ucp1 expression in differentiated primary adipocytes from sWAT of control and AdR2KO mice. After differentiation for 8 days, mature adipocytes were treated with or without 10 μM isoproterenol (Iso), 1 μM AS1842856, or 50 μM SP600125 for 6 h n = 5–6 wells in (I); n = 3–8 wells in (J). (K) The mRNA levels of Mapk8 (encodes JNK1), Foxo1 , Ppargc1a , and Ucp1 in mature mouse adipocyte cells, 3T3-L1. Adipocytes were transfected with siRNAs for 72 h and then treated with 10 μM isoproterenol (Iso) for 6 h ( n = 6 wells per group). (L) The mRNA levels of Foxo1 , Ppargc1a , and Ucp1 in differentiated mouse primary adipocytes from sWAT of control and AdR2KO mice. Adipocytes were transduced with low ( Foxo1L ) and high ( Foxo1H ) MOI of AAV-8 containing mouse Foxo1 plasmid for 7 days and then treated with 20 μM isoproterenol (Iso) for 16 h ( n = 8 wells per group). The immunoblot images are representative of at least three independent experiments. p values were determined by the two-tailed Student’s t test (A and F) or one-way ANOVA with Tukey’s multiple comparisons test (B-E and G-L). All data are presented as the mean ± SEM of at least three independent biological replicates. See also and . AS: AS1842856; SP: SP600125.

Article Snippet: anti-Phospho-Ser 256 FOXO1 , Cell Signaling Technology , Cat# 9461; RRID: AB_329831.

Techniques: Gene Expression, Western Blot, Expressing, Control, Transfection, Transduction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 deletion decreased the oxygen consumption rate in AdR2KO mice after cold exposure The whole-body oxygen consumption rate (VO 2 ), carbon dioxide production rate (VCO 2 ), and respiratory exchange rate (RER) in 10- to 12-week-old male control and AdR2KO mice housed at (A) room temperature (RT) or (B) cold (4 °C) for 72 h ( n = 6 mice per group). (C) The mRNA expression of brown fat-selective genes ( Ppargc1a , Ucp1 , and Cidea ) and general adipocyte genes ( Adipoq and Slc2a4 ) in the sWAT of male control and adipocyte-specific constitutively active ROCK (AdcaRK) mice ( n = 6 mice per group). (D) Immunohistochemistry for UCP1 in sections of sWAT from control and AdcaRK mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed squares. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. The images are representative of three independent experiments. (E) The mRNA level of Ucp1 in sWAT harvested from control and AdcaRK mice and then treated with 40 μM isoproterenol (Iso) and 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 6 wells per group). (F) The mRNA levels of Foxo1 and Irf4 in sWAT from male control and AdcaRK mice housed at RT or cold for 3 days ( n = 6 mice per group). p values were determined by two-tailed Mann-Whitney U tests (A and B) or one-way ANOVA with Tukey’s multiple comparisons test (C, E, and F). All data are presented as the mean ± SEM of at least three independent biological replicates. See also . AS: AS1842856.

Article Snippet: anti-Phospho-Ser 256 FOXO1 , Cell Signaling Technology , Cat# 9461; RRID: AB_329831.

Techniques: Control, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: DHA Increases Angptl4 Gene Expression and Reduces LPL Activity in a PPARγ-dependent manner in Adipocytes

doi: 10.1101/2025.11.15.685896

Figure Lengend Snippet: Markers of insulin signaling in mature 3T3-L1 adipocytes treated with different fatty acids. (A) The relative abundance of total FOXO1 protein. (B) The relative ratio of pFOXO1 ser 256 to total FOXO1 under basal (non-insulin stimulated) conditions. (C) The fold-change in the ratio of pFOXO1 to total FOXO1 and (D) The fold-change in the ratio of pAKT ser 473 to total AKT between basal and insulin stimulated conditions; The dotted line at y=1 represents the phosphorylation level of the basal conditions for each treatment. (E-F) representative blots, (-) indicates basal, (+) indicates insulin stimulated. Data are presented as mean ± SD (n = 6 passages of cells). Data were analyzed by repeated measures one-way ANOVA followed by a Tukey’s post-hoc test.

Article Snippet: The following primary antibodies were used: α-tubulin (CAT#: ab7291, Abcam, 1:1000 dilution), total AKT (CAT# 9272S, Cell Signaling, 1:1000 dilution), phosphor-Ser 473 AKT (CAT# 9271S, Cell Signaling, 1:1000 dilution), total FOXO1 (CAT# 9454S, Cell Signaling, 1:1000 dilution), phospho-Ser 256 FOXO1 (CAT# 9461S, Cell Signaling, 1:1000 dilution), and LPL (CAT# AF7197, biotechne, 1:1000 dilution).

Techniques: Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Effect of Long-Term Supplementation with Acetic Acid on the Skeletal Muscle of Aging Sprague Dawley Rats

doi: 10.3390/ijms23094691

Figure Lengend Snippet: Long-term supplementation of acetic acid increases the phosphorylation of AMP-activated protein kinase (AMPK), Akt, forkhead box protein O (FOXO)1, and FOXO3a. AMPK and pAMPK ( A ), Akt and pAkt ( B ), FOXO1 and pFOXO1 ( C ), FOXO3a and pFOXO3a ( D ), and α-tubulin were analyzed in the soleus muscle of water and acetic acid groups. They were analyzed by Western blotting, as described in the Materials and Methods Section. Values represent means ± SE for 4–5 rats. * p < 0.05, ** p < 0.01, *** p < 0.001, statistically significant versus the value of water group. Results were analyzed with Student’s t -test.

Article Snippet: Primary antibodies against AMPKα (#2532), phosphorylated Thr-172 AMPKα (#2535), Akt (#9272), phosphorylated Ser-473 Akt (#4058), forkhead box protein O1 (FOXO1) (#2880), phosphorylated Ser-256 FOXO1 (#9461), FOXO3a (#12829), and phosphorylated Ser-253 FOXO3a (#13129) were purchased from Cell Signaling Technology (Danvers, MA, USA); MEF2A (sc-313), PGC-1α (sc-517380), and Sp1 (sc-14027) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and α-tubulin (#017-25031) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Techniques: Phospho-proteomics, Western Blot