Journal: bioRxiv

Article Title: Direct mitochondrial import of lactate supports resilient carbohydrate oxidation

doi: 10.1101/2024.10.07.617073

Figure Lengend Snippet: a, qPCR measurement of Slc16a1 (Mct1) gene expression in heart and liver lysates from control ( Mct1 fl/fl ) and cardiac-specific knockout ( Mct1 iCKO ) mice 4 weeks post-induction ( Mct1 fl/fl n=8, Mct1 iCKO n=7 ). All data represent mean ± SEM. Significance determined by two-way ANOVA with Tukeys multiple comparison. b, Representative immunoblot analysis of MCT1, LDHA, LDHB, citrate synthase, calreticulin, and LDHD protein in heart lysates and purified mitochondria from Mct1 fl/fl and Mct1 iCKO mice 4 weeks post-induction. c, Schematic of the experimental design. Mitochondria were purified from human patient cardiac tissue, proteins were extracted from these mitochondria and immunoprecipitated by either an MCT1 antibody or an anti-IgG antibody. Eluted immunoprecipitants were run on a SDS Page gel and bands at the correct size were cut out and sent for proteomic analysis. d , Representative image of Coomassie blue stained gel of two unique human hearts, and e , corresponding immunoblot of MCT1 of the IP experiment. f , Sequence coverage (peptides) of human MCT1 from LC-MS/MS measurements of protein lysates from IP pulldown in b. g, Expression of SLC16A1 transcripts from single-nuclei data from donor hearts mapped to cell types. Data replotted from reference 25.

Article Snippet: The membrane was blocked with 5% non-fat milk (Serva) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) overnight at 4°C and then incubated for 3 hrs to overnight in 5% non-fat milk or 5% bovine serum albumin (Sigma) in TBS-T with primary antibodies against MPC1 (Cell Signaling, 1:1000), MPC2 (Cell Signaling, 1:1000), VDAC (Cell Signaling, 1:5000), MCT1 (Santa Cruz, 1:500, Proteintech, 1:1000), LDHA (Proteintech, 1:1000), LDHB (Proteintech, 1:1000), COX IV (Cell Signaling, 1:1000), ATP5A (Cell Signaling, 1:1000), or TOM20 (Cell Signaling, 1:1000).

Techniques: Expressing, Control, Knock-Out, Comparison, Western Blot, Purification, Immunoprecipitation, SDS Page, Staining, Sequencing, Liquid Chromatography with Mass Spectroscopy

Journal: Neurobiology of aging

Article Title: Prostaglandin J2 promotes O -GlcNAcylation raising APP processing by α and β-secretases: relevance to Alzheimer’s disease

doi: 10.1016/j.neurobiolaging.2017.10.009

Figure Lengend Snippet: Neurons were treated with DMSO (vehicle, control) or PGJ2 (10 μM) alone or in combination with Epoxomicin (5 nM, Epox, proteasome inhibitor), calpeptin (20 μM, Cpt, calpain inhibitor), pan caspase inhibitor (20 μM, Casp), and chloroquine (10 μM, CQ, lysosomal inhibitor). For comparison, neurons were also treated with each inhibitor alone. Total neuronal lysates were analyzed by western blotting (30μg of protein/lane) probed with the respective antibodies to detect in (A) full length APP (FL-APP, 22C11 antibody) and β-tubulin (loading control), and in (B) full length APP and its C-terminal fragment (FL-APP, 110kDa, APP-CTF, 15kDa, with the anti-CTF antibody) and actin (loading control). Molecular mass markers in kDa are shown in the center (A) and on the right (B). In (A) APP levels were semi-quantified by densitometry. Data in graphs represent the percentage of the pixel ratio for each of the three APP forms corresponding to mature (top and middle bands) and immature (bottom band) APP over β-tubulin for each condition compared to control (100%). Values are means ± s.e. from three independent experiments. Asterisks identify the values that are significantly different from PGJ2-treatment alone (** p<0.01; *** p<0.001). FL-APP, full length APP695; N, N-glycosylated; O, O-glycosylated; ns, non-significant.

Article Snippet: Inhibitors : epoxomicin (Peptides International Inc., Louisville, KY); calpeptin (Z-Leu-Nle-CHO), pan caspase inhibitor (Z-VAD-FMK), β-secretase inhibitor 2 (Z-VLL-CHO, BACE1-2) and 4 (BACE1-4), and tunicamycin (Calbiochem/EMD Bioscience, Gibbstown, NJ); chloroquine and brefeldin A (Sigma, St. Louis, MO); α-secretase inhibitor TAPI-2 and γ-secretase inhibitor BMS 299897 (Santa Cruz Biotechnology, Dallas, Tx).

Techniques: Control, Comparison, Western Blot

Journal: Neurobiology of aging

Article Title: Prostaglandin J2 promotes O -GlcNAcylation raising APP processing by α and β-secretases: relevance to Alzheimer’s disease

doi: 10.1016/j.neurobiolaging.2017.10.009

Figure Lengend Snippet: Neurons were treated as follows: (A) with DMSO (vehicle, control) or PGJ2 (10 μM) alone or in combination with tunicamycin (2 μM, Tunic, N-glycosylation inhibitor), or brefeldin A (15 μM, Bref. A, ER to Golgi protein transport inhibitor). For comparison, neurons were also treated with each drug alone. In (B) with DMSO (vehicle, control) or PGJ2 (10 μM), and the resulting neuronal lysates (30μg) were then treated with DMSO or with an O-deglycosylation mix containing neuroaminidase and O-glycosidase. In (C) with increasing concentrations of PGJ2 for 16h. In (D) with DMSO (vehicle, control), PGJ2 (10 μM), β-secretase inhibitor 2 (1 μM, BACE1-2), epoxomicin (5 nM, Epox, proteasome inhibitor), calpeptin (20 μM, Cpt, calpain inhibitor), or chloroquine (10 μM, CQ, lysosomal inhibitor) alone. Total neuronal lysates were analyzed by western blotting (30μg of protein/lane) probed with the respective antibodies to detect in: (A and B) full length APP (FL-APP, 22C11 antibody) and β-tubulin (loading control). In (C and D) O-GlcNAc and actin (loading control). Molecular mass markers in kDa are shown at the right. In (A and B) APP levels, and in (C and D) O-GlcNAcylated protein levels, were semi-quantified by densitometry. For (A and B), data in graphs represent the percentage of the pixel ratio for each of the three APP forms corresponding to mature (top and middle bands) and immature (bottom band) APP (unglycosylated APP also in A) over β-tubulin for each condition compared to control (100%). Values are means ± s.e. from four (A) and three (B) independent experiments. For (C and D) data in graphs represent the percentage of the pixel ratio for the levels of O-GlcNAcylated proteins (O-GlcNac/actin) over actin for each condition compared to control (100%). Values are means ± s.e. from four (C) and three (D) independent experiments. Asterisks identify the values that are significantly different from the control, (* p<0.05; ** p<0.01; *** p<0.001). FL-APP, full length APP695; N, N-glycosylated; O, O-glycosylated; O-GlcNAc, O-linked β-N-acetylglucosamine; unglyc., unglycosylated APP; ns, non-significant.

Article Snippet: Inhibitors : epoxomicin (Peptides International Inc., Louisville, KY); calpeptin (Z-Leu-Nle-CHO), pan caspase inhibitor (Z-VAD-FMK), β-secretase inhibitor 2 (Z-VLL-CHO, BACE1-2) and 4 (BACE1-4), and tunicamycin (Calbiochem/EMD Bioscience, Gibbstown, NJ); chloroquine and brefeldin A (Sigma, St. Louis, MO); α-secretase inhibitor TAPI-2 and γ-secretase inhibitor BMS 299897 (Santa Cruz Biotechnology, Dallas, Tx).

Techniques: Control, Glycoproteomics, Comparison, Western Blot

Journal: Oncology Letters

Article Title: Inhibition of NEDD8 and FAT10 ligase activities through the degrading enzyme NEDD8 ultimate buster 1: A potential anticancer approach

doi: 10.3892/ol.2016.5232

Figure Lengend Snippet: First- and second-generation proteasome inhibitors.

Article Snippet: Calpeptin (IPSI-001) , Lanospharma Laboratories Co., Ltd. (Chongqing, China) , Phase I.

Techniques: