FOXO3A Antibody - #AF6020

Fig. 4. Knockdown of FOXO3a compromised the inhibitory effects of TMF on C/EBPβ-mediated ECM degradation. (A–B) IHC assays were used to detect the altered expression of FOXO3a. (C–D) The protein expression of FOXO3a in shRNA-FOXO3a-infected C28/I2 cells was detected by western blotting assays. (E–K) Western blotting assays were used to detect the protein expression of Aggrecan, ADAMTS5, C/EBPβ, p-C/EBPβ, caspase3, cleaved-caspase3, and FOXO3a in LV-sh-FOXO3a-infected C28/I2 cells treated with IL-1β. (L-M) The apoptosis rate was determined by a flow cytometer.

Figure 10 Bexarotene activates TFE3 through the AMPK-mTOR and AMPK-SPK2- CARM1 signaling pathways in SCI. (A) Western blot assay of the cytoplasmic expression levels of AMPK, p-AMPK, mTOR, and p-mTOR. (B) Quantification of AMPK, p-AMPK, mTOR, and p-mTOR from A, normalized to GADPH. (C) Western blot assay of the nuclear expression levels of AMPK, p-AMPK, FOXO3a, p-FOXO3a, SKP2, and CARM1. (D) Quantification of AMPK, p-AMPK, FOXO3a, p-FOXO3a, SKP2, and CARM1 from C, normalized to H3. (E, F) Immunoprecipitation images showing nuclear CARM1–TFE3 binding. Data are expressed as the mean ± SEM (n = 6 per group). **P < 0.01, vs. SCI group; ##P < 0.01, vs. SCI + Bex group. One-way analysis of variance with least significance difference post hoc test. (p-)AMPK: (phospho-) adenosine 5′-monophosphate-activated protein kinase; (p-)FOXO3a: (phospho-)forkhead box O3; (p-)mTOR: (phospho-)mammalian target of rapamycin; Bex: bexarotene; CARM1: coactivator-associated arginine methyltransferase 1; CC: compound C; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H3: histone protein H3; SCI: spinal cord injury; SKP2: S-phase kinase-associated protein 2; TFE3: transcription factor E3.

Figure 10 Bexarotene activates TFE3 through the AMPK-mTOR and AMPK-SPK2- CARM1 signaling pathways in SCI. (A) Western blot assay of the cytoplasmic expression levels of AMPK, p-AMPK, mTOR, and p-mTOR. (B) Quantification of AMPK, p-AMPK, mTOR, and p-mTOR from A, normalized to GADPH. (C) Western blot assay of the nuclear expression levels of AMPK, p-AMPK, FOXO3a, p-FOXO3a, SKP2, and CARM1. (D) Quantification of AMPK, p-AMPK, FOXO3a, p-FOXO3a, SKP2, and CARM1 from C, normalized to H3. (E, F) Immunoprecipitation images showing nuclear CARM1–TFE3 binding. Data are expressed as the mean ± SEM (n = 6 per group). **P < 0.01, vs. SCI group; ##P < 0.01, vs. SCI + Bex group. One-way analysis of variance with least significance difference post hoc test. (p-)AMPK: (phospho-) adenosine 5′-monophosphate-activated protein kinase; (p-)FOXO3a: (phospho-)forkhead box O3; (p-)mTOR: (phospho-)mammalian target of rapamycin; Bex: bexarotene; CARM1: coactivator-associated arginine methyltransferase 1; CC: compound C; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H3: histone protein H3; SCI: spinal cord injury; SKP2: S-phase kinase-associated protein 2; TFE3: transcription factor E3.

FIGURE 5 FMN inhibits myostatin-mediated dephosphorylation on the PI3K/Akt/FoxO3a signalling pathway in the muscle of CKD rats and C2C12 myotubes. (A) Protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in gastrocnemius muscle analysed using Western blotting (n = 3/group). (B) Protein levels of MAFbx and MuRF-1 in gastrocnemius muscles analysed using Western blotting (n = 3/ group). (C) C2C12 myotubes were treated with FMN (50 μmol/L) in the presence or absence of TNF-α (40 ng/mL) for 48 h following 24 h of incubation with si-myostatin or si-NC. The myotubes were divided into four groups: si-NC, si-NC + TNF-α, si-NC + TNF-α + FMN (50 μmol/L) and si-myostatin + TNF-α. Protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in C2C12 myotubes (n = 3/ group). (D) Protein levels of MAFbx and MuRF-1 in C2C12 myotubes (n = 3/group). (E) Myostatin OE transfection was used to overexpress myostatin in C2C12 myotubes, and they were incubated with FMN (50 μmol/L) and TNF-α for another 48 h. The myotubes were divided into four groups: vector NC, vector NC + TNF-α, vector NC + TNF-α + FMN (50 μmol/L) and myostatin OE + TNF-α + FMN (50 μmol/L). The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in the myotubes were analysed using Western blotting. (F) Protein levels of MAFbx and MuRF1 in C2C12 myotubes analysed by Western blotting (n = 3/group). *P < .05, **P < .01

FIGURE 5 CTSS deficiency ameliorated stress-related metabolic molecular alterations. (A) Representative immunoblotting images and quantitative data for p-PI3K, p-Akt, p-FoxO3ɑ, MAFbx, MuRF-1, PI3K, Akt, FoxO3ɑ and GAPDH in gastrocnemius muscles at Day 14 after stress (n = 4). Data are mean ± SEM, and p-values were determined by a one-way ANOVA followed by Bonferroni post hoc tests. CW: CTSS+/+ control mice; CK: CTSS−/− control mice; SW: 14-day-stressed CTSS+/+ mice; SK: 14-day-stressed CTSS−/− mice. *p