epitope directed anti slc1a5  (Alomone Labs)

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: P2X7 Receptor Augments LPS-Induced Nitrosative Stress by Regulating Nrf2 and GSH Levels in the Mouse Hippocampus

doi: 10.3390/antiox11040778

Figure Lengend Snippet: Primary antibodies and lectin used in the present study.

Article Snippet: ASCT2 , Rabbit , Alomone labs (Jerusalem, Israel) (#ANT-082) , 1:500 (WB).

Techniques: Plasmid Preparation

Journal: Antioxidants

Article Title: P2X7 Receptor Augments LPS-Induced Nitrosative Stress by Regulating Nrf2 and GSH Levels in the Mouse Hippocampus

doi: 10.3390/antiox11040778

Figure Lengend Snippet: Effects of P2X7R deletion on GSH concentration and expressions of GCLC, GSHS, GS, GLS, ASCT2 and xCT following LPS injection. Under physiological condition, P2X7R deletion reduces GSH level in the hippocampus. However, P2X7R deletion increases GS and ASCT2 levels. LPS declines GSH concentration in P2X7R +/+ mice more than in P2X7R −/− mice. LPS decreases GSHS, GS and ASCT2 levels, but increases GLS and xCT levels only in the P2X7R +/+ mice. ( A ) Total GSH level in the hippocampus under physiological and post-LPS treated conditions. ( B ) Representative Western blot of GCLC, GSHS, GS, GLS, ASCT2 and xCT in the whole hippocampi of that P2X7R +/+ and P2X7R −/− mice. ( C – G ) Quantification of GSHS, GS, GLS, ASCT2 and xCT levels based on Western blot data. Open circles indicate each individual value. Horizontal and error bars indicate the mean value and S.E.M., respectively (*, # p < 0.05 vs. control and WT mice, n = 7, respectively). Full-length gel images of Western blot data in ( B ) could be found in .

Article Snippet: ASCT2 , Rabbit , Alomone labs (Jerusalem, Israel) (#ANT-082) , 1:500 (WB).

Techniques: Concentration Assay, Injection, Western Blot

Journal: Antioxidants

Article Title: P2X7 Receptor Augments LPS-Induced Nitrosative Stress by Regulating Nrf2 and GSH Levels in the Mouse Hippocampus

doi: 10.3390/antiox11040778

Figure Lengend Snippet: Effects of P2X7R deletion on ASCT2 expression and its S-nitrosylation following LPS injection. Under physiological conditions, total ASCT2 level in P2X7R −/− mice is higher than that of P2X7R +/+ mice. However, SNO-ASCT2 level in P2X7R −/− mice is lower than that of P2X7R +/+ mice. LPS decreases total ASCT2 level but increases SNO-ASCT2 level in P2X7R +/+ mice. LPS also increases SNO-ASCT2 level in P2X7R −/− mice without affecting total ASCT2 level. Total- and SNO-ASCT2 levels show a direct proportional relationship with Total- and SNO-GS levels in P2X7R +/+ and P2X7R −/− mice, respectively. ( A ) Representative Western blot of total- and SNO- ASCT2 in the whole hippocampi of that P2X7R +/+ and P2X7R −/− mice. ( B ) Quantification of total- and SNO-ASCT2 level based on Western blot data. Open circles indicate each individual value. Horizontal and error bars indicate the mean value and S.E.M., respectively (* p < 0.05 vs. WT mice, n = 7, respectively). ( C ) Representative Western blot of total- and SNO-ASCT2 in the whole hippocampi of that P2X7R +/+ mice following LPS treatment. ( D ) Quantification of total- and SNO-ASCT2 level based on Western blot data (* p < 0.05 vs. control mice, n = 7, respectively). ( E ) Representative Western blot of total- and SNO-ASCT2 in the whole hippocampi of that P2X7R −/− mice following LPS treatment. ( F ) Quantification of total- and SNO-ASCT2 level based on Western blot data (* p < 0.05 vs. control mice, n = 7, respectively). ( G ) Linear regression analyses of total- and SNO proteins between ASCT2 and GS in P2X7R +/+ and P2X7R −/− mice. Full-length gel images of Western blot data in this figure could be found in .

Article Snippet: ASCT2 , Rabbit , Alomone labs (Jerusalem, Israel) (#ANT-082) , 1:500 (WB).

Techniques: Expressing, Injection, Western Blot