Journal: bioRxiv

Article Title: A cell-autonomous mechanism regulates BCAA catabolism in white adipocytes and systemic metabolic balance

doi: 10.1101/2023.07.31.551146

Figure Lengend Snippet: A : Schematic of the major neutral amino acid transporters involved in cellular BCAA uptake. Asct2 (Slc1a5) transports glutamine into the cell establishing a chemical gradient that drives the uptake of BCAAs. In response to an increase in intracellular levels of glutamine, CD98 (Slc7a5), which exists as a heterodimer with Lat1 (Slc7a5), exports glutamine out of the cell while Lat1 (Slc7a5) simultaneously imports BCAAs. B : Relative gene expression of neutral amino acid transporters Asct2, CD98, and Lat1 in WAT of lean 4-month-old control and AnkB-RW mice. C : Representative immunoblots showing expression of AnkB and amino acid transporters in WAT of lean 4-month-old control mice. D : Relative expression of AnkB and amino acid transporters normalized to total protein (Revert 700, Ponceau S) or tubulin levels. E : Quantification of leucine uptake by differentiated primary white adipocytes. For all panels data show mean ± SEM with dots representing individual values. Data in B and D was collected from 3-4 mice per genotype. Data in E shows six biological replicates from two independent experiments. Data are reported as the mean ± SEM. Data was analyzed by an unpaired t test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: We also used rabbit anti-α-tubulin (11224-1-AP), rabbit anti Fabp4 (12802-1-AP), and mouse anti-GAPDH (2D4, 60004-I-AP) from Proteintech; rabbit anti-ASCT2 (ANT-082) from Alomone; and rabbit antibodies against GAPDH (D16H11 XP) and ACL (4332) from Cell Signaling Technologies.

Techniques: Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: A cell-autonomous mechanism regulates BCAA catabolism in white adipocytes and systemic metabolic balance

doi: 10.1101/2023.07.31.551146

Figure Lengend Snippet: A-C : Representative immunoblots showing expression of neutral amino acid transporters Asct2, CD98 and Lat1 in liver (A) , skeletal muscle (B) , and BAT (C) of lean 4-month-old control and AnkB-RW mice. D-F : Relative expression of indicated amino acid transporters normalized to total protein (Revert 700, Ponceau S). Data was collected from 4 mice per genotype. For all panels data show mean ± SEM with dots representing individual values. Data was analyzed by an unpaired t test. ns p >0.05.

Article Snippet: We also used rabbit anti-α-tubulin (11224-1-AP), rabbit anti Fabp4 (12802-1-AP), and mouse anti-GAPDH (2D4, 60004-I-AP) from Proteintech; rabbit anti-ASCT2 (ANT-082) from Alomone; and rabbit antibodies against GAPDH (D16H11 XP) and ACL (4332) from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: A cell-autonomous mechanism regulates BCAA catabolism in white adipocytes and systemic metabolic balance

doi: 10.1101/2023.07.31.551146

Figure Lengend Snippet: A : Relative gene expression of Asct2 in WAT of lean 3-month-old control and AT-AnkB KO mice. B : Representative immunoblots showing expression of Asct2 in WAT of lean 3-month-old mice. C : Relative Asct2 expression normalized to total protein (Revert 520) levels. For all panels data show mean ± SEM with dots representing individual values. Data was collected from 3 mice per genotype. Data was analyzed by an unpaired t test. **p < 0.01.

Article Snippet: We also used rabbit anti-α-tubulin (11224-1-AP), rabbit anti Fabp4 (12802-1-AP), and mouse anti-GAPDH (2D4, 60004-I-AP) from Proteintech; rabbit anti-ASCT2 (ANT-082) from Alomone; and rabbit antibodies against GAPDH (D16H11 XP) and ACL (4332) from Cell Signaling Technologies.

Techniques: Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: A cell-autonomous mechanism regulates BCAA catabolism in white adipocytes and systemic metabolic balance

doi: 10.1101/2023.07.31.551146

Figure Lengend Snippet: A : Schematic depicting full-length (FL), membrane binding domain (MBD), and ZU5-C-terminal (ZU5-Ct) AnkB constructs used to evaluate AnkB interaction with Asct2. B : Representative immunoblots showing expression of GFP-AnkB proteins and mCherry-Asct2 in total lysates and immunoprecipitation (IP) eluates. C : Representative immunofluorescent images show Asct2 (magenta) distribution in differentiated white adipocytes at the basal state and upon BCAA treatment. BODIPY (green) labels lipid droplets. DAPI (bue) labels nuclei. D : Quantification of surface Asct2 in control (untreated n=14, BCAA-treated n=15) and AnkB-RW (untreated n=31, BCAA-treated n=17) white adipocytes. E : Representative immunoblots showing surface levels of indicated proteins in control and AnkB-RW cultured white adipocytes. F : Quantification of surface levels of Asct2, Na + /K + ATPAse, Fabp4, and Lat1 normalized to total surface protein levels (Revert 520) collected from three independent experiments. For all panels data show mean ± SEM. Data in D was analyzed by One-way ANOVA with Tukey’s post hoc analysis test for multiple comparisons. Data in F was analyzed by an unpaired t test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns p >0.05.

Article Snippet: We also used rabbit anti-α-tubulin (11224-1-AP), rabbit anti Fabp4 (12802-1-AP), and mouse anti-GAPDH (2D4, 60004-I-AP) from Proteintech; rabbit anti-ASCT2 (ANT-082) from Alomone; and rabbit antibodies against GAPDH (D16H11 XP) and ACL (4332) from Cell Signaling Technologies.

Techniques: Membrane, Binding Assay, Construct, Western Blot, Expressing, Immunoprecipitation, Control, Cell Culture

Journal: Antioxidants

Article Title: Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis

doi: 10.3390/antiox13070771

Figure Lengend Snippet: Increased EAAC1 gene and protein expression in Neuro2a cells with Erdr1 siRNA knockdown. ( a ) Confocal images showing the effect of transfection with a specific siRNA targeting Erdr1 (siRNA) on the intensity of EAAC1 expression in Neuro2a cells compared with that in the negative control cells (scrambled siRNA). Scale bar, 100 μm. Insets in the panels are enlarged images corresponding to the box highlighted on the full images. Scale bar, 20 μm. ( b ) Quantification of relative EAAC1 densities in Neuro2a cells is shown. n = 5; 5–6 areas were measured in each sample. ** p < 0.01. ( c ) Protein expression of EAAC1 in Neuro2a cells transfected with a specific siRNA targeting Erdr1 (siRNA) and in the negative control cells (scrambled siRNA). Quantification of the EAAC1 protein expression (data in the left panel) by densitometry is shown in the right panel. n = 6. * p < 0.05.

Article Snippet: Non-specific staining was blocked with the reagent PBS containing 5% BSA, and the cells were incubated with anti-EAAC1 (Alomone Labs) at 1:1000 dilution for 1 h. After a wash with PBS-Tween20, the cells were labeled with fluorescent-labeled secondary antibodies Alexa-Fluor 488 anti-rabbit IgG (Invitrogen) at 1:1000 dilutions for 30 min.

Techniques: Expressing, Knockdown, Transfection, Negative Control

Journal: Antioxidants

Article Title: Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis

doi: 10.3390/antiox13070771

Figure Lengend Snippet: The schematic illustration indicates the relationship between Erdr1, GTRAP3-18, EAAC1, and GSH in the brain/hippocampus and Neuro2a/primary hippocampal neurons. In the brain/hippocampus of GTRAP3-18-deficient mice, GTRAP3-18 may be regulated by Erdr1 and exert compensatory feedback on Erdr1 expression. In Neuro2a/primary hippocampal neurons, knockdown of Erdr1 resulted in a decrease in GTRAP3-18, upregulation of EAAC1 expression, leading to an increase in GSH levels.

Article Snippet: Non-specific staining was blocked with the reagent PBS containing 5% BSA, and the cells were incubated with anti-EAAC1 (Alomone Labs) at 1:1000 dilution for 1 h. After a wash with PBS-Tween20, the cells were labeled with fluorescent-labeled secondary antibodies Alexa-Fluor 488 anti-rabbit IgG (Invitrogen) at 1:1000 dilutions for 30 min.

Techniques: Expressing, Knockdown

Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

Article Title: Glutamatergic Dysfunction of Astrocytes in Paraventricular Nucleus of Thalamus Contributes to Adult Anxiety Susceptibility in Adolescent Ethanol Exposed Mice

doi: 10.1038/s41386-025-02264-3

Figure Lengend Snippet: (a) The quantification of glutamate levels in the dorsal thalamic area including PVT. (b-d) Representative expression of GLT1 (as known as EAAT2), GLAST (as known as EAAT1), and GAPDH (b) and pooled data (c-d) of western blots showing that the GLT1 expression in the PVT of AIE mice is selectively reduced compared to that of the CON mice. (e-f) Representative figures (e) and pooled data (f) confirming the reduction of GLT1 in the conditional GLT1 knockdown mice (GLT1 cHET ). (g-h) Representative figures (g) and pooled data (h) showing the selective reduction of GLT1 in astrocytes, not in neurons of PVT. (i-k) Non-invasive magnetic resonance spectroscopy (MRS) measurement showing the increase in glutamate levels in the dorsal thalamic area including PVT of GLT1 cHET . (l-q) Representative traces (l, o) and pooled data (m-n, p-q) showing the anxiogenic profiles of GLT1 cHET in the open field test (l-n) and elevated plus maze test (o-q). Female (circles) and male (triangles). Data represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 3i , 3l , and 3o were created using BioRender.com .

Article Snippet: These membranes were incubated with anti-GLT1 antibody (1:2000, Guinea pig, Millipore Sigma Cat# AB1783, RRID:AB_90949), anti-GLAST antibody (1:2000, Rabbit, Alomone Labs Cat# AGC-021, RRID:AB_2039885), anti-GAPDH antibody (1:2000, Mouse, Sigma Aldrich Cat# MAB374, RRID:AB_2107445), and anti-xCT antibody (1:1000, Rabbit, ABclonal Cat# A2413, RRID:AB_2863004) overnight at 4 °C.

Techniques: Expressing, Knockdown, Western Blot, Spectroscopy

Journal: Molecular Medicine Reports

Article Title: Reoxygenation induces reactive oxygen species production and ferroptosis in renal tubular epithelial cells by activating aryl hydrocarbon receptor

doi: 10.3892/mmr.2020.11679

Figure Lengend Snippet: AhR activation status does not affect Nrf2 activation or transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blots of Nrf2 levels (corresponding to its activation status) and the expression of the Nrf2 transcriptional targets xCT (SLC7A11) and SOD-3. (B-D) Statistical analysis of the western blots. Neither Reox nor CH223191 affects Nrf2 activity, or the expression of xCT and SOD-3. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; xCT, cystine-glutamate antiporter; SOD-3, superoxide dismutase; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Article Snippet: Primary antibodies were specific for AhR (1:200; cat. no. sc-133088; Santa Cruz Biotechnology, Inc.), cytochrome P450 family 1 subfamily A member 1 (CYP1A1; 1:500; cat. no. sc-25304; Santa Cruz Biotechnology, Inc.), Nrf2 (1:1,000; cat. no. TA343586; OriGene Technologies, Inc.), superoxide dismutase 3 (SOD-3; 1:100; cat. no. sc-271170; Santa Cruz Biotechnology, Inc.), cystine-glutamate antiporter (xCT, also known as SLC7A11; 1:1,000; cat. no. ANT-111; Alomone Labs), HIF-1α (1:500; cat. no. sc-10790; Santa Cruz Biotechnology, Inc.), LDH-A (1:1,000; cat. no. 2012; Cell Signaling Technology, Inc.), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Activity Assay, Cell Culture, Western Blot, Expressing