Journal: Redox biology

Article Title: The E3 ubiquitin ligase TRIM31 is involved in cerebral ischemic injury by promoting degradation of TIGAR.

doi: 10.1016/j.redox.2021.102058

Figure Lengend Snippet: Fig. 4. TRIM31 deficiency maintained mitochondria homeostasis in brain of mice subjected to middle cerebral artery occlusion (MCAO). (A) TRIM31 deficiency improved ultrastructural defects of neuronal mitochondria from mice subjected to MCAO. (B–D) The effect of TRIM31 on the expression of protein related to mitochondrial dynamics, PGC-1α, MFN1, MEN2 and DRP1, in the mice brain after 24 h of MCAO. Scale bars: 10 μm. Data were presented as the mean ± SEM of 3 independent experiments. *p < 0.05 vs. indicated group.

Article Snippet: Primary antibodies against TRIM31 (12543-1-AP), G6PD (66 373-1-1 g), DRP1 (12957-1-AP), MFN1 (13798-1-AP), MFN2 (12186-1-AP) and PGC-1α (66 369-1-1 g) were from Proteintech (Wuhan, China).

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Adropin attenuates pancreatitis-associated lung injury through PPARγ phosphorylation-related macrophage polarization

doi: 10.3892/ijmm.2023.5298

Figure Lengend Snippet: Excessive M1 macrophage polarization is observed in the Adro-KO + L-Arg group. (A) Western blot analysis of lung tissue form the Adro-KO + L-Arg group. (B) Co-expression of CD68 and iNOS in lung tissue in L-Arg group determined using immunofluorescence. (C) Co-expression of CD68 and CD206 of lung tissue in the L-Arg group determined using immunofluorescence. (D) iNOS mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥8). (E) CD68 mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥7). (F) CD163 mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥8). (G) Arg-1 mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥8). (H) Quantitative analysis of immunofluorescence (CD68 + iNOS) staining (n≥5). (I) Quantitative analysis of immunofluorescence (CD68; CD206) staining in macrophages (n≥9). (J) Quantitative analysis of the western blots (PPARγ; n≥6); (K) Quantitative analysis of the western blots (PPARγ Ser273; n≥6). (L) Quantitative analysis of the western blots (PPARγ Ser112; n≥6). * P<0.05. L-Arg, L-arginine; NS, normal saline; Adro-KO, adropin knockout; iNOS, inducible nitric oxide synthase.

Article Snippet: The primary antibodies were used at 4°C overnight, and the antibody included Adropin (1:1,000, cat. no. PA5-72781, Thermo Fisher Scientific, Inc.); GADPH (1:10,000; cat. no. AC001, ABclonal); peroxisome proliferator-activated receptor γ (PPARγ; 1:1,000, cat. no. bsm-52220R, BIOSS); p-PPARγ Ser112 (1:1,000, cat. no. bs-3737R, BIOSS); p-PPARγ Ser273 (1:1,000, cat. no. bs-2875R, BIOSS), caspase-3 (1:1,000, cat. no. YC0006, ImmunoWay Biotechnology Company) and PARP1 (1:1,000, cat. no. A0942, ABclonal).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Saline, Knock-Out

Journal: International Journal of Molecular Medicine

Article Title: Adropin attenuates pancreatitis-associated lung injury through PPARγ phosphorylation-related macrophage polarization

doi: 10.3892/ijmm.2023.5298

Figure Lengend Snippet: Adropin exogenous supplement induces M2 macrophage polarization. (A) Western blot analysis of lung tissue from the Adro-KO + L-Arg + Adro (34−76) group. (B) Quantitative analysis of the western blots (PPARγ, PPARγ Ser112, PPARγ Ser273) (n≥5). (C) Co-expression of CD68 and iNOS in lung tissue from the Adro-KO + L-Arg+Adro (34−76) group. (D) Co-expression of CD68 and iNOS in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group. (E) Ratio of CD68/DAPI of lung in Adro-KO+ L-Arg+Adro (34−76) group(n≥3); (F) The ratio of iNOS/DAPI in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥3). (G) Ratio of iNOS/CD68 in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥3). (H) Ratio of CD68/DAPI in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥3). (I) Ratio of CD206/DAPI in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥3). (J) Ratio of CD206/CD68 in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥3). (K) CD163 mRNA level in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥6). (L) Arg-1 mRNA level in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥6). (M) iNOS mRNA level in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥6). (N) CD86 mRNA level level in lung tissue from the Adro-KO + L-Arg + Adro (34−76) group (n≥6). * P<0.05 and *** P<0.001. L-Arg, L-arginine; Adro-KO, adropin knockout; NS, normal saline; iNOS, inducible nitric oxide synthase; Arg-1, arginase 1.

Article Snippet: The primary antibodies were used at 4°C overnight, and the antibody included Adropin (1:1,000, cat. no. PA5-72781, Thermo Fisher Scientific, Inc.); GADPH (1:10,000; cat. no. AC001, ABclonal); peroxisome proliferator-activated receptor γ (PPARγ; 1:1,000, cat. no. bsm-52220R, BIOSS); p-PPARγ Ser112 (1:1,000, cat. no. bs-3737R, BIOSS); p-PPARγ Ser273 (1:1,000, cat. no. bs-2875R, BIOSS), caspase-3 (1:1,000, cat. no. YC0006, ImmunoWay Biotechnology Company) and PARP1 (1:1,000, cat. no. A0942, ABclonal).

Techniques: Western Blot, Expressing, Knock-Out, Saline

Journal: International Journal of Molecular Sciences

Article Title: Effects of Maternal Resveratrol on Maternal High-Fat Diet/Obesity with or without Postnatal High-Fat Diet

doi: 10.3390/ijms21103428

Figure Lengend Snippet: Primary and secondary antibodies used for Western blot.

Article Snippet: pPPARγ (phosphoryl peroxisome proliferator-activated receptors γ) , Bioss , bs-3737R , 1:1000.

Techniques: Western Blot

Journal: Open Life Sciences

Article Title: Sini Decoction Intervention on Atherosclerosis via PPARγ-LXRα-ABCA1 Pathway in Rabbits

doi: 10.1515/biol-2018-0053

Figure Lengend Snippet: Expression profiling of PPARγ-LXRα signaling to confirm the mechanism of treating AS by SND. The PPARγ (A-D) and LXRα (E-H) proteins levels in liver, peritoneal macrophages, PMC, and adipose tissue were measured by western blotting. The relative quantities were analyzed by ImageJ software. Data shown as mean ± SEM (n = 6). # P<0.05, compared with Chow group; *P<0.05, compared with HFD group.

Article Snippet: Primary antibodies to ABCA1 and Apo-B were purchased from Abcam (MA, USA); PPARγ antibody and LXRα antibody were purchased from Proteintech (IL, USA); and ApoA-I antibody was purchased from MyBioSource (CA, USA).

Techniques: Expressing, Western Blot, Software

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 1: PGC-1b expression is downregulated in skeletal muscle upon fasting. (A) Gene expression of PGC-1b and PGC-1a relative to TATA-box binding protein (TBP) in Quadriceps muscle of sedentary or exercised mice. (B and C) Gene expression of PGC-1b and PGC-1a relative to 18S in liver (B) and Gastrocnemius muscle (C) of ad-libitum fed or 24 h fasted mice. (D) Gene expression of PGC-1b relative to TATA-box binding protein (TBP) in different muscles and other tissues of control (WT) and PGC-1b muscle-specific knockout (MKO) mice. (E) Gene expression of PGC-1b, mitochondrial target genes and PGC-1a relative to TATA-box binding protein (TBP) in Gastrocnemius muscle of WT and MKO mice. (F and G) Protein levels of different mitochondrial chain complexes (F) in Gastrocnemius muscle of WT and MKO mice and representative immunoblots (G). As a loading control eukaryotic elongation factor 2 (eEF2) was used. (H and I) Quantification of (H) and representative succinate dehydrogenase (SDH) and cytochrome oxidase (COX) stainings of Gastrocnemius muscle cryo-sections (I) of WT and MKO mice. * indicates significant differences between sedentary and exercised mice, fed and fasted mice and WT and MKO mice; n ¼ 3e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Expressing, Gene Expression, Binding Assay, Muscles, Control, Knock-Out, Western Blot

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 3: PGC-1b is necessary for the fasting-induced fiber atrophy. (AeC) Gastrocnemius (A), Soleus (B) and Tibialis anterior (TA) (C) absolute and relative muscle weights of ad-libitum fed or 24 h fasted mice. D-G) Minimal fiber ferrets (minFerret) of oxidative (D and E) and glycolytic (F and G) Gastrocnemius muscle cross-sections of ad-libitum fed (D and F) or 24 h fasted (E and G) mice. (H) Representative images of Gastrocnemius muscle cross-sections of ad-libitum fed or 24 h fasted WT and MKO mice. * indicates significant differences between WT and MKO mice; # indicates significant differences between fed and fasted conditions; n ¼ 4e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques:

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 4: Transcriptomic patterns in MKO link modulated muscle proteostasis to fiber atrophy. (A) Gene expression changes in fed and fasted WT and MKO animals. A selection of Annotation Clusters indicates major predicted functional pathways that are altered only in the comparison of fed, in the overlap, and only in the comparison of fasted mice, respectively. (B) Gene expression comparison of the fasting response in WT and MKO mice. A selection of Annotation Clusters indicates major predicted functional pathways of physiological fasting that are dependent on muscle PGC-1b (WT only) and independent of muscle PGC-1b (overlap). The pathways found only in the comparison of MKO mice depict changes that arise de novo due to the absence of muscle PGC-1b in fasting. (C) Heatmaps of the relative expression of genes involved in protein synthesis and ribosome (left panel), and protein ubiquitination and proteasome (right panel). Colors represent the log2 fold change in gene expression compared to WT fed animals. (D) Gene expression of endogenous PGC-1b and PGC-1a relative to 18S in primary myotubes of WT mice treated with forskolin, 8-(4-chlorophenylthio)adenosine 30,50-cyclic monophosphate (CPT-cAMP) or 3-isobutyl-1-methylxanthine (IBMX) for 6 h (n ¼ 3). (E) Protein levels of overexpressed, epitope-tagged (HA) PGC-1b, endogenous p-CREBS133, endogenous CREB and endogenous eEF2 in primary myotubes of WT mice treated with forskolin for 6 h (n ¼ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Gene Expression, Selection, Functional Assay, Comparison, Expressing, Ubiquitin Proteomics

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 5: Fasted MKO mice show reduced induction of myostatin, atrophy marker gene expression and protein ubiquitination. (A) Gene ontology (GO) analysis of differentially expressed (DE) genes between fasted MKO vs. fasted WT mice. (B) Gene expression of PGC-1b, myostatin (Mstn), muscle RING finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx) relative to 18S in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice (n ¼ 5e6). (C and D) Representative immunoblot of ubiquitinylated proteins (C) in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice and corresponding quantification (D). Equal loading was verified by Ponceau staining of the membranes. (E) Gene expression of forkhead box O (Foxo) transcription factors 1, 3 and 4 relative to 18S in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice. (F and G) Representative immunoblots of total and phosphorylated forkhead box O 3a (Foxo3a) proteins in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice and corresponding quantifications (G). As a loading control eukaryotic elongation factor 2 (eEF2) was used. * indicates significant differences between WT and MKO mice; # indicates significant differences between fed and fasted conditions; n ¼ 3e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Marker, Gene Expression, Ubiquitin Proteomics, Western Blot, Staining, Control

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 7: Nfatc1 activity is increased in fasted MKO animals. (A) ISMARA predictions of enriched transcription factor binding motifs in the comparison of fed and fasted WT and MKO mice, and the fasting response in WT and MKO animals. (B) Nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1) is the top ISMARA predicted motif from the gene expression profiles obtained in Gastrocnemius muscle of 24 h fasted WT and MKO mice. (C) Gene expression of Nfatc1 and predicted Nfatc1 target genes PGC-1a, protein-O-mannose kinase (Pomk), membrane associated ring-CH-type finger 1 (March1), SH3 domain containing kinase binding protein 1 (Sh3kbp1), methyltransferase like 11B (Mettl11b) and nitric oxide synthase 1 (Nos1) relative to 18S in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice. (D) Reporter gene assay using a 3 NFAT-luc plasmid, and co-transfection of NFATC1 alone or together with PGC-1b overexpression plasmids. (E and F) Representative immunoblots of total and phosphorylated Ca2þ/calmodulin-dependent protein kinase IIa (CaMKIIa) protein levels in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice (E) and corresponding quantifications (F). As a loading control eukaryotic elongation factor 2 (eEF2) was used. * indicates significant differences between WT and MKO mice; # indicates significant differences between fed and fasted conditions; n ¼ 3e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Activity Assay, Binding Assay, Comparison, Gene Expression, Membrane, Reporter Gene Assay, Plasmid Preparation, Cotransfection, Over Expression, Western Blot, Control