Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Blueprint of the distinct metabolite profiles of healthy pig heart chambers ☆

doi: 10.1016/j.jmccpl.2025.100462

Figure Lengend Snippet: Protein turnover-related metabolites, such as amino acids and dipeptides are differently abundant in the heart chambers. ( A ) The heatmap shows the distribution of dipeptides in the heart chambers (n = 30 per group). (*, p < 0.05), (**, q < 0.05) in ANOVA, unadjusted and adjusted. ( B–D ) An abundance of amino acids varies between chambers (n = 30 per group). ( E ) The ratio between phosphorylated p70S6k and p70S6k. ( F ) mTor intensity in the chambers. ( G ) Western blot analysis of ribosomal protein kinase S6, mammalian target of rapamycin, and their phosphorylated forms in chambers ( n = 6 per group). Legend: ( B–F ) (*, p < 0.05), (**, p < 0.01), (***, p < 0.001), and (****, p < 1e−04) in t -test, unadjusted. LV = left ventricle, RV = right ventricle, LA = left atrium, RA = right atrium, mTor = mammalian target of rapamycin, p70S6K = ribosomal protein kinase S6.

Article Snippet: Proteins were transferred to the nitrocellulose membrane and incubated overnight at 4 °C with primary antibodies against the mammalian target of rapamycin (mTOR) (Cell Signaling #2972), phospho-mTOR (Cell Signaling #5536), ribosomal protein S6 kinase beta-1 (p70 S6K) (Cell Signaling #9202), and phospho-p70 S6K (Cell Signaling #9205).

Techniques: Western Blot

Journal: Nature cell biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources.

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: Fig. 1 | Blockage of lysosomal function disconnects mTORC1 localization and substrate-specific activity. a, A schematic model of the pharmacological inhibition of lysosomal function by BafA1 targeting the v-ATPase. b,c, Basal lysosomal proteolysis in HEK293FT cells shown by accumulation of LC3B upon BafA1 treatment (100 nM, 6 h before fixation) (b) and quantification of LC3B signal (c). nCtrl = 49 and nBafA1 = 50 individual cells from five independent fields per condition. d, A schematic representation of the treatment strategy followed in this study, assessing mTORC1 activity under basal (unchallenged cells), starvation or acute re-activation (AA add-back) conditions. AA levels are shown by a black line, and mTORC1 activity by a red line (see also Methods). e,f, Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, treated as indicated, using confocal microscopy (magnified insets shown on the right; scale bars, 25 μm and for insets, 5 μm) (e) and quantification of colocalization (f). n = 50 individual cells from five independent fields per condition. g, Immunoblots with lysates from HEK293FT WT cells treated with

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Activity Assay, Inhibition, Activation Assay, Marker, Confocal Microscopy, Western Blot

Journal: Nature cell biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources.

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: Fig. 2 | Blockage of lysosomal protease activity disconnects mTORC1 localization on lysosomes and activity towards cytoplasmic substrates. a, A schematic model of the pharmacological inhibition of lysosomal proteases by PepA and E64 blocking local AA production. b,c, Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells (b) and its quantification (c), treated as indicated, using confocal microscopy. PepA (50 μM) and E64 (25 μM) (or DMSO as control, Ctrl) were added directly in the media for 16 h before fixation (magnified insets shown to the right; scale bars, 25 μm and for insets, 5 μm). n = 56 individual cells from three independent fields per condition. Data shown as mean ± s.e.m. ****P < 0.0001. d, Immunoblots with lysates from HEK293FT WT cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and protease inhibitors

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Activity Assay, Inhibition, Blocking Assay, Marker, Confocal Microscopy, Control, Western Blot

Journal: Nature cell biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources.

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: Fig. 4 | The persistent phosphorylation of cytoplasmic mTORC1 substrates in cells with non-lysosomal mTOR is not due to impaired or slower substrate dephosphorylation. a,b, mTOR/LAMP2 colocalization (a) and its quantification (b). mTOR delocalizes away from lysosomes already after 2 h of BafA1 treatment. Time course of BafA1 treatment (100 nM, 2–8 h) to block lysosomal function in HEK293FT cells. Magnified insets shown to the right (a). Scale bars, 25 μm and insets, 5 μm. n = 49–50 individual cells from five independent fields per condition (see also Methods). c–e, Dephosphorylation kinetics of lysosomal (TFEB) and cytoplasmic (S6K and 4E-BP1) substrates of mTORC1 upon BafA1 treatment (100 nM, 1–8 h) in HEK293FT cells showing a rapid drop in TFEB

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Blocking Assay

Journal:

Article Title: Mapping key functional sites within yeast TFIID

doi: 10.1038/sj.emboj.7600111

Figure Lengend Snippet: Analysis of a recombinant complex containing seven TAFs. (A) Seven TAFs (hTAF4, 5, 6, 9, 10, 12 and mTAF8) were coexpressed in Sf9 cells and the TAF10-containing complexes were purified using a TAF10-specific antibody. The bound complexes were eluted and separated by gel filtration on a Superdex 200 column. (A) SDS–PAGE analysis and silver staining of the collected fractions showing the formation of a homogeneous complex in fraction 14 with an estimated molecular weight of 800 kDa. The elution of molecular weight standards is indicated (top). (B) Western blot analysis showing that the seven TAFs coelute in the peak fractions of the Superdex 200 gel filtration profile. (C) Western blot analysis of anti-TAF10 immunoprecipitation (IP) experiments. Lane 1, coexpression of the seven TAFs; lane 2, TAF5 is missing in the coexpression; lane 3, TAF10 is missing in the coexpression. (D) Electron microscopic observation of the peak fraction 14 reveals a homogeneous population of complexes in negative stain. (E) Image analysis of the seven-TAF complex reveals a TFIID-like trilobed structure. The bar represents 50 nm in (D) and 13.5 nm in (E).

Article Snippet: Altogether, these results show that TAF5 is present as two copies in the complex, that the C-terminal part of the subunit is located in lobes A and B and suggest that the N-terminal ends of both copies are located in lobe C. Thus, TAF5 appears to be organized in a symmetric way and could form the linking regions between lobes A and C and between lobes B and C. Analysis of a recombinant TAF-containing complex To further investigate the putative architectural role of TAF5, different combinations of recombinant mammalian TAFs were coexpressed in Sf9 cells using a baculovirus expression system.

Techniques: Recombinant, Purification, Filtration, SDS Page, Silver Staining, Molecular Weight, Western Blot, Immunoprecipitation, Staining

Journal:

Article Title: Mapping key functional sites within yeast TFIID

doi: 10.1038/sj.emboj.7600111

Figure Lengend Snippet: Summary of the immunolabelling experiments. Proposed location of TAFs within the 3-D model of yTFIID. The location of histone fold-containing TAF pairs is schematically represented by green circles as determined previously (Leurent et al, 2002). The extended TAF1 and TAF5 subunits are schematically represented to highlight their presence in two lobes, Note that the size and shape of the coloured areas does not directly correspond to polypeptide mass.

Article Snippet: Altogether, these results show that TAF5 is present as two copies in the complex, that the C-terminal part of the subunit is located in lobes A and B and suggest that the N-terminal ends of both copies are located in lobe C. Thus, TAF5 appears to be organized in a symmetric way and could form the linking regions between lobes A and C and between lobes B and C. Analysis of a recombinant TAF-containing complex To further investigate the putative architectural role of TAF5, different combinations of recombinant mammalian TAFs were coexpressed in Sf9 cells using a baculovirus expression system.

Techniques: