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