Journal: Diabetes

Article Title: Illumination of the Endogenous Insulin-Regulated TBC1D4 Interactome in Human Skeletal Muscle

doi: 10.2337/db21-0855

Figure Lengend Snippet: Insulin-stimulated phosphorylation of the TBC1D1-TBC1D4 complex after exercise. IP of TBC1D1 from rested ( R ) and prior exercised ( E ) human skeletal muscle at basal ( B ) and in response to insulin ( I ) stimulation, followed by Western blot of TBC1D4 total ( A ), pTBC1D4 Ser318 ( B ), pTBC1D4 Thr642 ( C ), and pTBC1D4 Ser704 ( D ). E : Representative Western blot of IP data. Data were analyzed by repeated-measures ANOVA. ANOVA significance is displayed with text on each graph. Values for each individual are marked with a unique asterisk and connected with lines within each leg ( n = 7).

Article Snippet: Primary antibodies against pTBC1D4-Ser318 (8619), pTBC1D4-Thr642 (8881), pTBC1D1-Thr596 (6927S), pAkt-Thr308 (9275), pAkt-Ser473 (4271L), Akt2 (3063), pAMPK-Thr172 (2531), and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Western Blot

Journal: Diabetes

Article Title: Illumination of the Endogenous Insulin-Regulated TBC1D4 Interactome in Human Skeletal Muscle

doi: 10.2337/db21-0855

Figure Lengend Snippet: Insulin-stimulated regulation of the TBC1D1-TBC1D4 complex in skeletal muscle from healthy, lean individuals and obese individuals with type 2 diabetes. IP of TBC1D1 from basal and insulin-stimulated human skeletal muscle in healthy, lean individuals and obese individuals with type 2 diabetes, followed by Western blot of p-TBC1D4 Ser318 ( A ), p-TBC1D4 Thr642 ( B ), TBC1D1 total ( C ) and TBC1D4 ( D ). E : Ratio of TBC1D4 co-IP to TBC1D1 IP. Representative Western blot of IP data. Bars are means and lines represent individual responses ( n = 10). Data were analyzed by two-way repeated-measures ANOVA within group. ANOVA significance is displayed with text on each graph. Šidák multiple comparisons test was used as post hoc test. **** P < 0.0001, ** P < 0.01, and * P < 0.05.

Article Snippet: Primary antibodies against pTBC1D4-Ser318 (8619), pTBC1D4-Thr642 (8881), pTBC1D1-Thr596 (6927S), pAkt-Thr308 (9275), pAkt-Ser473 (4271L), Akt2 (3063), pAMPK-Thr172 (2531), and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Western Blot, Co-Immunoprecipitation Assay

Journal: Diabetes

Article Title: Illumination of the Endogenous Insulin-Regulated TBC1D4 Interactome in Human Skeletal Muscle

doi: 10.2337/db21-0855

Figure Lengend Snippet: Intact insulin- and exercise-mediated phosphorylation of TBC1D4 in complex with TBC1D1. IP of TBC1D1 from basal ( B ) and insulin (I)-stimulated human skeletal muscle, followed by Western blot of TBC1D4 total ( A ), pTBC1D4 Ser318 ( B ), and pTBC1D4 Thr642 ( C ). IP of TBC1D1 from rested (R) and acutely exercised ( E ) human skeletal muscle, followed by Western blot of TBC1D4 total ( D ), pTBC1D4 Ser318 ( E ), and pTBC1D4 Thr642 ( F ). G : Representative Western blot of IP data. Input refers to muscle lysate prior to IP. Dots and lines represent individual responses ( n = 11 in A – C and n = 8 D-F). Data were analyzed by paired Student t test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: Primary antibodies against pTBC1D4-Ser318 (8619), pTBC1D4-Thr642 (8881), pTBC1D1-Thr596 (6927S), pAkt-Thr308 (9275), pAkt-Ser473 (4271L), Akt2 (3063), pAMPK-Thr172 (2531), and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Berberine regulates mesangial cell proliferation and cell cycle to attenuate diabetic nephropathy through the PI3K/Akt/AS160/GLUT1 signalling pathway

doi: 10.1111/jcmm.17167

Figure Lengend Snippet: The RT‐qPCR primer sequences (Mouse)

Article Snippet: Anti‐PI3K‐p85 (#4292), anti‐Akt (4691S), anti‐p‐Akt (#4060), anti‐AS160 (#2670), anti‐p‐AS160 (#8619) and anti‐GAPDH (#5174) antibodies were obtained from Cell Signaling Biotechnology.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Berberine regulates mesangial cell proliferation and cell cycle to attenuate diabetic nephropathy through the PI3K/Akt/AS160/GLUT1 signalling pathway

doi: 10.1111/jcmm.17167

Figure Lengend Snippet: Effect of BBR on regulating the PI3K/Akt/AS160/GLUT1 pathway. Protein was extracted from cultured GMCs. GAPDH was used as internal controls. Intensity of PI3K, AKT, p‐AKT, AS160, p‐AS160, Total GLUT1 and m‐GLUT1 was quantified, normalized to internal control and expressed as mean ± SEM. (A,B) The levels of PI3K, Akt, p‐Akt, AS160, p‐AS160, Total GLUT1 and m‐GLUT1 were detected in each group; (C) RT‐qPCR analyses were performed to detect the mRNA expression of PI3K, Akt, AS160 and GLUT1 in each group. Relative expression of PI3K, Akt, AS160 and GLUT1. Data are expressed as Mean ± SEM, n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HG group. HG: high glucose; BBR: HG + BBR (60 μmol/L); LY294002 (HG + LY294002); Metformin: HG + metformin (5 mmol/L)

Article Snippet: Anti‐PI3K‐p85 (#4292), anti‐Akt (4691S), anti‐p‐Akt (#4060), anti‐AS160 (#2670), anti‐p‐AS160 (#8619) and anti‐GAPDH (#5174) antibodies were obtained from Cell Signaling Biotechnology.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing

Journal: Molecular cell

Article Title: Lysosome Positioning Influences mTORC2 and AKT Signaling

doi: 10.1016/j.molcel.2019.05.009

Figure Lengend Snippet: (A) WT, myrlysin-KO, myrlysin-rescue (myrlysin-KO transfected with myrlysin cDNA), diaskedin-KO, lyspersin-KO and KIF1B-KIF5B-KO HeLa cells were incubated in medium depleted of both amino acids and serum for 30 min, or serum for 1 h, and then refed with the corresponding nutrients for the indicated periods. Cell lysates were subjected to SDS-PAGE and immunoblotting with antibodies to phosphorylated TSC2 (p-TSC2), and total TSC2 (t-TSC2). Arrowheads indicate the specific p-TSC2 bands. (B) Quantification of the ratio of p-TSC2 to t-TSC2 upon combined amino-acid and serum depletion-refeeding. (C) Quantification of the ratio of p-TSC2 to t-TSC2 upon serum depletion-refeeding. The line graphs for WT and myrlysin-KO cells are the same shown in Fig. 3D. In B and C, values are the mean ± SEM from three independent experiments such as that shown in A. *p<0.01, **p<0.001, ***p<0.0001 (WT vs. myrlysin KO), two-way ANOVA with Tukey’s multiple comparisons test. (D) WT and myrlysin-KO HeLa cells were subjected to serum starvation and refeeding. Cell lysates were analyzed as in A for phosphorylated p-TSC2, t-TSC2, phosphorylated FoxO3 (p-FoxO3), total FoxO3 (t-FoxO3), phosphorylated AS160 (p-AS160), total AS160 (t-AS160), phosphorylated GSK-3β (p- GSK-3β), total GSK-β3 (p-GSK-3β), S473-phosphorylated AKT [p-AKT (S473)] and total AKT (t-AKT). In A and E, the positions of molecular mass markers (in kDa) are indicated on the left.

Article Snippet: Phospho-AS160 (Ser318) , Cell Signaling Technology , 8619.

Techniques: Transfection, Incubation, SDS Page, Western Blot, Serum Depletion

Journal: Molecular cell

Article Title: Lysosome Positioning Influences mTORC2 and AKT Signaling

doi: 10.1016/j.molcel.2019.05.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: Phospho-AS160 (Ser318) , Cell Signaling Technology , 8619.

Techniques: Recombinant, Electron Microscopy, Protease Inhibitor, Software, Modification, Transfection, Magnetic Beads

Journal: Physiological Reports

Article Title: Metformin does not compromise energy status in human skeletal muscle at rest or during acute exercise: A randomised, crossover trial

doi: 10.14814/phy2.14307

Figure Lengend Snippet: (a–c) Muscle Akt pSer308: Akt2 protein (a), and Akt pThr473: Akt2 protein (b), TBC1D1 pSer237: TBC1D1 protein (c), TBC1D1 pSer700: TBC1D1 protein (d), TBC1D4 pSer318: TBC1D4 protein (e), TBC1D4 pSer588: TBC1D4 protein (f) and TBC1D4 pThr642: TBC1D4 protein (g) in the short‐term study. Data are means ± SEM . Black bars shows results for the metformin trial. White bars shows results for the placebo trial. Representative immunoblots are shown in figure (h). ∗ p < .05 and ∗∗∗ p < .001 versus “Pre”; ††† p < .001 versus preceding value

Article Snippet: The ACC phosphospecific antibody is raised against a peptide corresponding to the sequence in rat ACC1 containing the Ser79 phosphorylation site, but the antibody also recognized the human ACC1 and ACC2 when phosphorylated most likely at the corresponding Ser80 and Ser221, respectively (Abu‐Elheiga, Almarza‐Ortega, Baldini & Wakil, ); ACC protein content (Dako, Streptavidin/HRP P0397); Akt2 protein content (Cell Signalling Technology Inc, #3063); Akt phosphorylation at Thr308 (Cell Signalling Technology Inc, #9275); Akt phosphorylation at Ser473 (Cell Signalling Technology Inc, #9271); TBC1D1 protein (provided by Prof Hardie DG, University of Dundee, UK); TBC1D1 phosphorylation at Ser237 (Millipore, #2061452); TBC1D1 phosphorylation at Ser700 – (Cell Signalling Technology Inc, #9271); TBC1D4 protein content (Abcam, #24469); TBC1D4 phosphorylation at Ser318 (Cell Signalling Technology Inc, #8619); TBC1D4 phosphorylation at Ser588 (Cell Signalling Technology Inc, #8730); TBC1D4 phosphorylation at Thr642 (Symansis, NZ #3028 P1).

Techniques: Western Blot