anti tcp1  (Proteintech)

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: TCP1 is positively related to the tumorigenesis and development of AML. ( A ) In the successive generations, the tumor formation rate increased in the HL-60 cell line. ( B ) Mass spectrometry combined with two-dimensional in-gel electrophoresis revealed that TCP1 is a highly oncogenic gene. ( C ) Differentially expressed protein spots marked with master numbers displayed in 2D-DIGE images. The arrows indicate the 33 significantly upregulated protein spots (marked with master numbers) and 54 downregulated protein spots (marked with master numbers) displayed in 2D-DIGE images. ( D ) Proteomic analysis of HL-60 cells using 2D-DIGE. The arrows indicate protein spots that differed significantly between the HL-60 cells and the control cells. Relative spot intensity was calculated on the basis of the spot volume. Only the spots indicated with arrows were used for identification. ( E ) IHC staining examined TCP1 expression in tumor tissues of different generations. The TCP1 expression in tissue samples of different stage of AML patients was detected by RT-qPCR ( F ) and Western blotting ( G ). ( H ) Western blotting was used to examine the expression of TCP1 in different leukemia cell lines. Data are presented as the means ± SDs from three independent experiments. ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Mass Spectrometry, Nucleic Acid Electrophoresis, Control, Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: The effect of TCP1 on AML cell development and proliferation in vitro and in vivo. ( A ) The efficiency of shTCP1 was measured in HL-60-G4 cells by Western blotting. ( B ) A clonogenic cell survival assay was performed to evaluate the growth of HL-60-G4-shTCP1 cells in vitro. ( C , D ) Representative cell cycle distribution of HL-60-G4 cells after knocking down TCP1 with lentivirus transfection. ( E ) Detection of xenograft tumor formation in nude mice using Tet-on technology. ( F ) Representative image of Dox- and Dox+ groups of tumors at the end of the experiment. ( G ) Western blotting was used to analyze the protein level of TCP1 in tumor tissues in each group. Data are presented as the means ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: In Vitro, In Vivo, Western Blot, Clonogenic Cell Survival Assay, Transfection

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: TCP1 regulated the stability of PLK1 and AML1-ETO. ( A ) Western blotting analysis of TCP1, PLK1, and AML1-ETO expression in HL-60-G4-shTCP1 and Kasumi-1-shTCP1 cells. ( B ) Cells were incubated with 10 μM CHX alone or combined with 10 μM MG132, and WB assays were used to determine the levels for TCP1 and PLK1. ( C ) The image shown in B was quantified using ImageJ software (Version 1.8.0). ( D ) Co-IP assays were performed to observe the pull-down levels of PLK1 and AML1-ETO in the shTCP1 group and scramble group. Data are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Western Blot, Expressing, Incubation, Software, Co-Immunoprecipitation Assay

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: MiR-340-5P inhibits AML cell proliferation by silencing TCP1. ( A ) Dual-luciferase reporter system analysis of targeted transcriptional regulation of TCP1 by miRNAs. ( B ) HEK293 cells were co-transfected with miR-340-5p mimics or control and reporter plasmid or the mutant 3′-UTR of TCP1, and the luciferase activity was measured after 48 h. ( C ) MiR-340-5p levels in AML cell lines were tested by RT-qPCR. ( D ) HL-60-G4 cells overexpressing (O/E) the vector control or miR-340-5p. Detection of TCP1 expression in HL-60-G4 cells overexpressing miR-340-5p by Western blot. ( E ) The cellular proliferation of HL-60-G4 O/E cells was determined by cell counting. ( F ) HL-60-G4 O/E cells were treated with 100 nmol L −1 of an hsa-miR-340-5p inhibitor for 24 h, and cellular proliferation was examined. Data are presented as the means ± SDs from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Luciferase, Transfection, Control, Plasmid Preparation, Mutagenesis, Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Cell Counting

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: FTY720 has an affinity for TCP1 and can inhibit AML cell proliferation. ( A ) The chemical structure of FTY720. ( B ) TCP1 subunit protein and FTY720 molecular docking virtual screening. ( C ) Surface plasmon resonance detected binding between FTY720 and TCP1. ( D ) The effect of FTY720 on the viability of AML cells. ( E ) TCP1 protein levels in the three cell lines were examined by Western blotting and ( F ) normalized to the corresponding density of GAPDH. ( G ) The sensitivity of HL-60 cells to FTY720 increased as the TCP1 protein level increased. ( H ) HL-60-G4 cells formed many more colony units than HL-60 cells, and their colony numbers decreased as FTY720 increased. ( I ) The quantification and plot were created using ImageJ software (Version 1.8.0) for the image shown in H. Data are presented as the means ± SDs from three independent experiments. *** p < 0.001.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: SPR Assay, Binding Assay, Western Blot, Software

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: FTY720 induces cell cycle arrest and decreases protein levels by inhibiting TCP1 chaperone function in AML cells. ( A ) Representative cell cycle distribution images. ( B ) Column chart of the cell cycle ratio of HL-60-G4, HL-60, Kasumi-1, and KG1α cells. ( C ) Changes in intracellular protein levels in HL-60-G4 and KG1α cells incubated with FTY720 for 24 h. ( D ) The image shown in C was quantified using ImageJ software (Version 1.8.0). ( E ) The interaction between the PLK1 protein and TCP1 protein in HL-60-G4 and KG1α cells exposed to FTY720 for 24 h was assayed by Co-IP. ( F ) The interaction between the CDC20 protein and TCP1 protein in HL-60-G4 and KG1α cells exposed to FTY720 for 24 h was assayed by Co-IP. ( G ) Changes in intracellular protein levels in Kasumi-1 cells incubated with FTY720 for 24 h were detected by Western blotting. ( H ) TCP1 interacts with AML1-ETO in Kasumi-1 cells, but FTY720 disrupts the interaction. Means ± SDs of three independent experiments are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Incubation, Software, Co-Immunoprecipitation Assay, Western Blot

Journal: Cell reports

Article Title: Meteorin-like alleviates hepatic steatosis by regulating hepatic triglyceride secretion and fatty acid oxidation.

doi: 10.1016/j.celrep.2025.115246

Figure Lengend Snippet: Figure 4. Metrnl regulates PC biosynthesis and TG secretion via the Sp1/CCTa axis (A) The Kennedy pathway for PC biosynthesis. (B and C) FFA-treated HepG2 cells were transfected with an adenovirus for 48 h, and the mRNA levels of the genes involved in PC biosynthesis (B) and the protein levels of Sp1 and the genes involved in PC biosynthesis (C). (D and E) Protein levels in FFA-treated AML12 cells (D) and primary hepatocytes from LKO-Met mice and L-WT mice (E). (F–H) The expression levels were measured in the livers of HFD-fed and HFD-rMet mice using qPCR (F), WB (G), and IHC (H) (n = 5). (I) Sp1 expression was detected in the nuclear extracts of FFA-treated HepG2 cells 48 h after the adenovirus infection (n = 3). (J–L) Sp1 and CCTa expression (J) and PC (K) and TG contents (L) were determined in FFA-treated HepG2 cells transfected with the adenovirus and incubated with 100 nmol/L Mith (an Sp1-specific inhibitor) for 24 h (n = 3). (M) A dual-luciferase assay was performed in HEK293T cells 24 h after the transfection of a CCTa-promoter plasmid and adenovirus infection, and the empty plasmid pGL3 was used as a control (n = 3). (N) qPCR analysis of the levels of the CCTa promoter enriched with an anti-Sp1 antibody or an immunoglobulin G (IgG) antibody via a ChIP assay in Metrnl- overexpressing or control HepG2 cells. Mith, mithramycin. The data are presented as the mean ± SD. *p < 0.05 and **p < 0.01 compared with the Ad-Vec, sh-NC, or HFD groups.

Article Snippet: The proteins were subsequently transferred to PVDF membranes and incubated with primary antibodies against GAPDH, Metrnl, CCTa, CHKA, Sp1, phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK (Proteintech), CPT1A, phospho-LKB1 (Ser428), phospho-CaMKK2 (Ser495), and phospho-TAK1 (Thr184/187) overnight at 4 C. The membranes were washed before an incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Transfection, Expressing, Infection, Incubation, Luciferase, Plasmid Preparation, Control

Journal: Cell reports

Article Title: Meteorin-like alleviates hepatic steatosis by regulating hepatic triglyceride secretion and fatty acid oxidation.

doi: 10.1016/j.celrep.2025.115246

Figure Lengend Snippet: Figure 5. Metrnl activates Sp1/CCTa-mediated PC biosynthesis and TG secretion through the PI3K p110a/Akt pathway (A–C) The protein expression levels were measured using WB in FFA-treated HepG2 cells (A), AML12 cells (B), and primary hepatocytes isolated from L-WT and LKO-Met mice treated with rMet (100 ng/mL) (C) for 24 h or left untreated. (D–F) The protein expression levels (D), PC content (E), and TG secretion in the supernatant (F) were determined in FFA-treated HepG2 cells incubated with or without LY294002 (10 mmol/L) and the adenovirus for 24 h. (G–J) The Metrnl concentration in the medium was measured using ELISA (G), and Akt activity was confirmed by treating FFA-treated HepG2 cells with the Metrnl- neutralizing antibody (H) and measuring the PC content (I) and TG secretion in the medium (J) 24 h after the adenovirus infection. The isotype control antibody was used as a control antibody. (K–L) Protein levels were determined after treatment with different doses of rMet (K) with or without LY294002 (LY), PIK75 (PIK) (100 mmol/L), TGX221 (TGX) (100 mmol/L), or rMet (100 ng/mL) for 30 min (L) (n = 3). The data are presented as the mean ± SD. *p < 0.05 and **p < 0.01 compared with the Ad-Vec, sh-Vec, or Ctrl groups.

Article Snippet: The proteins were subsequently transferred to PVDF membranes and incubated with primary antibodies against GAPDH, Metrnl, CCTa, CHKA, Sp1, phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK (Proteintech), CPT1A, phospho-LKB1 (Ser428), phospho-CaMKK2 (Ser495), and phospho-TAK1 (Thr184/187) overnight at 4 C. The membranes were washed before an incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Expressing, Isolation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Infection, Control

Journal: Cell reports

Article Title: Meteorin-like alleviates hepatic steatosis by regulating hepatic triglyceride secretion and fatty acid oxidation.

doi: 10.1016/j.celrep.2025.115246

Figure Lengend Snippet: Figure 4. Metrnl regulates PC biosynthesis and TG secretion via the Sp1/CCTa axis (A) The Kennedy pathway for PC biosynthesis. (B and C) FFA-treated HepG2 cells were transfected with an adenovirus for 48 h, and the mRNA levels of the genes involved in PC biosynthesis (B) and the protein levels of Sp1 and the genes involved in PC biosynthesis (C). (D and E) Protein levels in FFA-treated AML12 cells (D) and primary hepatocytes from LKO-Met mice and L-WT mice (E). (F–H) The expression levels were measured in the livers of HFD-fed and HFD-rMet mice using qPCR (F), WB (G), and IHC (H) (n = 5). (I) Sp1 expression was detected in the nuclear extracts of FFA-treated HepG2 cells 48 h after the adenovirus infection (n = 3). (J–L) Sp1 and CCTa expression (J) and PC (K) and TG contents (L) were determined in FFA-treated HepG2 cells transfected with the adenovirus and incubated with 100 nmol/L Mith (an Sp1-specific inhibitor) for 24 h (n = 3). (M) A dual-luciferase assay was performed in HEK293T cells 24 h after the transfection of a CCTa-promoter plasmid and adenovirus infection, and the empty plasmid pGL3 was used as a control (n = 3). (N) qPCR analysis of the levels of the CCTa promoter enriched with an anti-Sp1 antibody or an immunoglobulin G (IgG) antibody via a ChIP assay in Metrnl- overexpressing or control HepG2 cells. Mith, mithramycin. The data are presented as the mean ± SD. *p < 0.05 and **p < 0.01 compared with the Ad-Vec, sh-NC, or HFD groups.

Article Snippet: The proteins were subsequently transferred to PVDF membranes and incubated with primary antibodies against GAPDH, Metrnl, CCTa, CHKA, Sp1, phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK (Proteintech), CPT1A, phospho-LKB1 (Ser428), phospho-CaMKK2 (Ser495), and phospho-TAK1 (Thr184/187) overnight at 4 C. The membranes were washed before an incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Transfection, Expressing, Infection, Incubation, Luciferase, Plasmid Preparation, Control

Journal: Cell reports

Article Title: Meteorin-like alleviates hepatic steatosis by regulating hepatic triglyceride secretion and fatty acid oxidation.

doi: 10.1016/j.celrep.2025.115246

Figure Lengend Snippet: Figure 5. Metrnl activates Sp1/CCTa-mediated PC biosynthesis and TG secretion through the PI3K p110a/Akt pathway (A–C) The protein expression levels were measured using WB in FFA-treated HepG2 cells (A), AML12 cells (B), and primary hepatocytes isolated from L-WT and LKO-Met mice treated with rMet (100 ng/mL) (C) for 24 h or left untreated. (D–F) The protein expression levels (D), PC content (E), and TG secretion in the supernatant (F) were determined in FFA-treated HepG2 cells incubated with or without LY294002 (10 mmol/L) and the adenovirus for 24 h. (G–J) The Metrnl concentration in the medium was measured using ELISA (G), and Akt activity was confirmed by treating FFA-treated HepG2 cells with the Metrnl- neutralizing antibody (H) and measuring the PC content (I) and TG secretion in the medium (J) 24 h after the adenovirus infection. The isotype control antibody was used as a control antibody. (K–L) Protein levels were determined after treatment with different doses of rMet (K) with or without LY294002 (LY), PIK75 (PIK) (100 mmol/L), TGX221 (TGX) (100 mmol/L), or rMet (100 ng/mL) for 30 min (L) (n = 3). The data are presented as the mean ± SD. *p < 0.05 and **p < 0.01 compared with the Ad-Vec, sh-Vec, or Ctrl groups.

Article Snippet: The proteins were subsequently transferred to PVDF membranes and incubated with primary antibodies against GAPDH, Metrnl, CCTa, CHKA, Sp1, phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK (Proteintech), CPT1A, phospho-LKB1 (Ser428), phospho-CaMKK2 (Ser495), and phospho-TAK1 (Thr184/187) overnight at 4 C. The membranes were washed before an incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Expressing, Isolation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Infection, Control

Journal: Cell reports

Article Title: Meteorin-like alleviates hepatic steatosis by regulating hepatic triglyceride secretion and fatty acid oxidation.

doi: 10.1016/j.celrep.2025.115246

Figure Lengend Snippet: Figure 6. Metrnl overexpression promotes hepatic FAO in an AMPK-dependent manner (A) The mRNA levels of genes involved in lipid metabolism in FFA-treated HepG2 cells (n = 3). (B–E) WB analysis of the AMP-activated protein kinase (AMPK) pathway in FFA-treated HepG2 cells 24 h after the adenovirus infection (B), in AML12 cells 24 h after rMet (100 ng/mL) treatment (C), in primary hepatocytes from L-WT and LKO-Met mice treated with rMet (100 ng/mL) for 24 h or left untreated (D), and in the livers of HFD and HFD-rMet mice (E) (n = 3–5). (F‒G) Hepatic b-hydroxybutyric acid (F) and ATP contents (G) were detected in HFD-fed and HFD-fed rMet mice (n = 5). (H–J) WB analysis (H) and measurement of the b-hydroxybutyric acid (I) and ATP contents (J) in FFA-treated HepG2 cells after adenovirus treatment with or without Com C challenge (10 mmol/L) for 24 h (n = 3).

Article Snippet: The proteins were subsequently transferred to PVDF membranes and incubated with primary antibodies against GAPDH, Metrnl, CCTa, CHKA, Sp1, phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK (Proteintech), CPT1A, phospho-LKB1 (Ser428), phospho-CaMKK2 (Ser495), and phospho-TAK1 (Thr184/187) overnight at 4 C. The membranes were washed before an incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Over Expression, Infection

Journal: Cell reports

Article Title: Meteorin-like alleviates hepatic steatosis by regulating hepatic triglyceride secretion and fatty acid oxidation.

doi: 10.1016/j.celrep.2025.115246

Figure Lengend Snippet: Figure 7. The restoration of PC biosynthesis and AMPK activation are sufficient to rescue hepatic TG accumulation in LKO-Met mice fed an HFD (A) Six-week-old LKO-Met and L-WT mice were fed an HFD for 12 weeks. In the last 2 weeks, the mice were injected with saline, CDP-choline (150 mg/kg), or AICAR (0.5 mg/g) for 2 weeks (n = 5). (B–F) TG secretion in the serum after fasting for 4 h and subsequent poloxamer 407 treatment (B), serum TG content (C), hepatic PC content (D), H&E staining (E), and the hepatic TG content (F) were determined. L-WT, Metrnlflox/flox,ALB Cre mice; LKO-Met, Metrnl flox/flox,ALB Cre+ mice; CDP, CDP-choline; AICAR, acadesine, an AMPK activator. The data are presented as the mean ± SD. **p < 0.01 and ***p < 0.001 compared with L-WT mice; #p < 0.05, ##p < 0.01, and ####p < 0.0001 for the comparison of LKO-Met+CDP-choline mice and LKO-Met mice.

Article Snippet: The proteins were subsequently transferred to PVDF membranes and incubated with primary antibodies against GAPDH, Metrnl, CCTa, CHKA, Sp1, phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK (Proteintech), CPT1A, phospho-LKB1 (Ser428), phospho-CaMKK2 (Ser495), and phospho-TAK1 (Thr184/187) overnight at 4 C. The membranes were washed before an incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Activation Assay, Injection, Saline, Staining, Comparison