antibody against total akt  (Cell Signaling Technology Inc)

Journal: The EMBO Journal

Article Title: YTHDC1 promotes postnatal brown adipose tissue development and thermogenesis by stabilizing PPARγ

doi: 10.1038/s44318-025-00460-x

Figure Lengend Snippet: ( A ) The YTHDC1 and GAPDH protein levels in iBATs of 16-week HFD-fed mice, 8-week-old db/db mice, and their respective control mice were measured by immunoblotting ( n = 4 per group). ( B ) The body weights of Ythdc1 -BKO and Ythdc1 flox/flox mice fed HFD diet for 17 weeks ( n = 12 per group). ( C , D ) The morphology of iBATs in 25-week-old HFD-fed mice. ( E ) The relative iBAT weight in 25-week-old HFD-fed mice ( n = 11 per group; P < 0.0001). ( F ) The relative liver weight in 25-week-old HFD-fed mice ( n = 11 per group; P = 0.003). ( G ) The morphology of liver and H&E staining of liver sections in 25-week-old HFD-fed mice. ( H ) Glucose tolerance tests (GTTs) were conducted in 24-week-old HFD-fed Ythdc1 flox/flox and Ythdc1 -BKO mice ( n = 8 per group; 0 min: P = 0.1244; 15 min: P = 0.0359; 30 min: P = 0.0016; 60 min: P = 0.0037; 120 min: P = 0.0023). ( I ) Insulin tolerance tests (ITTs) were performed in 24-week-old HFD-fed Ythdc1 flox/flox and Ythdc1 -BKO mice ( n = 8 per group; 0 min: P = 0.8149; 15 min: P = 0.0008; 30 min: P = 0.0098; 60 min: P = 0.0053). ( J ) The serum insulin levels of 25-week-old HFD-fed Ythdc1 flox/flox and Ythdc1 -BKO mice ( n = 12 per group; P = 0.0072). ( K , L ) Hepatic p-AKT (S473), p-AKT (T308), and AKT protein levels were measured by immunoblotting and quantified using ImageJ in 25-week-old HFD-fed Ythdc1 flox/flox and Ythdc1 -BKO mice ( n = 3 per group; p-AKT (S473) BKO VS flox/flox: P < 0.0001; p-AKT (T308) BKO VS flox/flox P < 0.0001). Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Studient’s t test analysis. Differences among more than two groups were analyzed by one-factor analysis of variance (ANOVA). * P < 0.05. ** P < 0.01. n was the number of biologically independent mice. .

Article Snippet: Livers were then isolated, homogenized in lysis buffer (R0020, Solarbio), and subjected to immunoblotting with antibodies against phosphorylated AKT (pSer473 and pThr308) and total AKT (Cell Signaling Technology).

Techniques: Control, Western Blot, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: TBK1 inhibitor amlexanox exerts anti-cancer effects against endometrial cancer by regulating AKT/NF-κB signaling

doi: 10.7150/ijbs.100212

Figure Lengend Snippet: TBK1 expression is correlated with the epithelial-mesenchymal transition (EMT)-related marker expression and cell migration in endometrial cancer. (A) Spearman correlation analysis between TBK1 and EMT-related gene expression in The Cancer Genomic Atlas (TCGA) endometrial cancer database. (B) Immunoblots of N-cadherin, vimentin, snail, and TBK1 in the cell lysates of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells treated with 10 ng/mL transforming growth factor (TGF)-β1 or vehicle. Band intensities were quantified and normalized to the control levels. (C) Wound-healing assay of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or vehicle. Images were taken 24 h after the scratch wound. (D) Quantification of cell migration expressed as a percentage of control values. (E) Transwell migration assay of lentiviral sh-TBK1- or sh-Luc-infected HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or vehicle. Images were taken after 24 h under a light microscope. Scale bars, 200 μm. (F) Quantification of cell migration expressed as a percentage of control values. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Immunoblotting and immunostaining were performed using antibodies against phosphorylated (p)-TBK1, total TBK1, N-cadherin, E-cadherin, snail, vimentin, p-AKT, total AKT, p-NF-κB, total NF-κB, p-p70S6K, total p70S6K (Cell Signaling Technology, Danvers, MA, USA), α-tubulin, and β-actin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: Expressing, Marker, Migration, Gene Expression, Western Blot, Infection, Control, Wound Healing Assay, Transwell Migration Assay, Light Microscopy

Journal: International Journal of Biological Sciences

Article Title: TBK1 inhibitor amlexanox exerts anti-cancer effects against endometrial cancer by regulating AKT/NF-κB signaling

doi: 10.7150/ijbs.100212

Figure Lengend Snippet: Amlexanox inhibits the migration of endometrial cancer cells. (A) Immunoblots of N-cadherin, vimentin, and snail in the cell lysates of HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or 100 μM amlexanox for 36 h. Band intensities were quantified and normalized to the control levels. (B) Wound-healing assay of HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or 100 μM amlexanox. Images were taken 24 h after the scratch wound. (C) Quantification of cell migration expressed as a percentage of control values. (D) Transwell migration assay of HEC-1A and Ishikawa cells treated with 10 ng/mL TGF-β1 or 100 μM amlexanox. Images were taken after 24 h under a light microscope. Scale bars, 200 μm. (E) Quantification of cell migration expressed as a percentage of control values. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Immunoblotting and immunostaining were performed using antibodies against phosphorylated (p)-TBK1, total TBK1, N-cadherin, E-cadherin, snail, vimentin, p-AKT, total AKT, p-NF-κB, total NF-κB, p-p70S6K, total p70S6K (Cell Signaling Technology, Danvers, MA, USA), α-tubulin, and β-actin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: Migration, Western Blot, Control, Wound Healing Assay, Transwell Migration Assay, Light Microscopy

Journal: International Journal of Biological Sciences

Article Title: TBK1 inhibitor amlexanox exerts anti-cancer effects against endometrial cancer by regulating AKT/NF-κB signaling

doi: 10.7150/ijbs.100212

Figure Lengend Snippet: Targeting TBK1 inhibits the proliferation and migration of endometrial cancer cells via the AKT/NF-κB pathway. (A) WST-8 assay of HEC-1A and Ishikawa cells treated with 100 μM MK-2206 or 100 μM amlexanox for 24 h. (B) Immunoblots of N-cadherin and snail in the cell lysates of HEC-1A and Ishikawa cells pretreated with 100 μM MK-2206 or 100 μM amlexanox for 36 h in the presence of 10 ng/mL TGF-β1. Band intensities were quantified and normalized to the control levels. (C) Wound-healing assay of HEC-1A and Ishikawa cells pretreated with 100 μM MK-2206 or 100 μM amlexanox in the presence of 10 ng/mL TGF-β1. Images were taken 24 and 48 h after the scratch wound. (D) Quantification of cell migration expressed as a percentage of control values. (E) Transwell migration assay of HEC-1A and Ishikawa cells pretreated with 100 μM MK-2206 or 100 μM amlexanox in the presence of 10 ng/mL TGF-β1. Images were taken after 24 h under a light microscope. Scale bars, 200 μm. (F) Quantification of cell migration expressed as a percentage of control values. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Immunoblotting and immunostaining were performed using antibodies against phosphorylated (p)-TBK1, total TBK1, N-cadherin, E-cadherin, snail, vimentin, p-AKT, total AKT, p-NF-κB, total NF-κB, p-p70S6K, total p70S6K (Cell Signaling Technology, Danvers, MA, USA), α-tubulin, and β-actin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: Migration, Western Blot, Control, Wound Healing Assay, Transwell Migration Assay, Light Microscopy

Journal: International Journal of Biological Sciences

Article Title: TBK1 inhibitor amlexanox exerts anti-cancer effects against endometrial cancer by regulating AKT/NF-κB signaling

doi: 10.7150/ijbs.100212

Figure Lengend Snippet: Targeting TBK1 inhibits tumor growth in vivo . (A) Representative images of subcutaneous xenograft tumors in nude mice treated with 5 mg/kg amlexanox or vehicle. Red arrows indicate the xenograft tumor. (B) Tumor growth curve in each group. (C) Tumor weights in each group. (D, E) RT-qPCR analysis of MKI67, CDKN1A , and CDKN1B mRNA levels in the xenograft tissues of each group. (F, G) Immunoblots of N-cadherin, vimentin, snail, p-AKT, AKT, p-NF-κB, and NF-κB in the xenograft tumor lysates of nude mice of each group. Band intensities were quantified and normalized to the control levels. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. (H) Schematic diagram showing the anti-cancer mechanism of TBK1 inhibition, leading to decreased tumor growth, migration, and EMT in endometrial cancer cells.

Article Snippet: Immunoblotting and immunostaining were performed using antibodies against phosphorylated (p)-TBK1, total TBK1, N-cadherin, E-cadherin, snail, vimentin, p-AKT, total AKT, p-NF-κB, total NF-κB, p-p70S6K, total p70S6K (Cell Signaling Technology, Danvers, MA, USA), α-tubulin, and β-actin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: In Vivo, Quantitative RT-PCR, Western Blot, Control, Inhibition, Migration