Journal: The Journal of Biological Chemistry

Article Title: Mapping the intracellular HMGB1 interactome and alterations induced by Toll-like receptor 4 activation

doi: 10.1016/j.jbc.2025.110866

Figure Lengend Snippet: The HMGB1 interactome in THP-1 cells changes after TLR4 activation. A , illustration of construct used to insert MycBioID2-HMGB1 into THP-1 cells. B , titration of doxycycline concentration to induce MycBioID2-HMGB1 expression. Lysates were analyzed by Western blotting using anti-Myc-HRP. GAPDH was used as loading control. C , Volcano plot of the HMGB1 interactome in resting (n = 3) and LPS-stimulated (n = 3) THP-1 cells. p -values were calculated by multiple paired two-way ANOVA corrected for multiple testing by FDR. D , the subcellular localization of proteins in the HMGB1 interactome. E , lysates from resting and LPS stressed THP-1 cells were fractioned into nuclear (N), cytosolic (C) and mitochondrial (M) fractions and analyzed by Western blotting using anti-HMGB1 and anti-BioID2. LaminB1, GAPDH and VDAC were used as loading controls (n = 3). All Western blots have been marked with molecular weights (kDa) on the right side.

Article Snippet: The following antibodies were used: 2040 (Mouse monoclonal-HRP against Myc-tag, Cell signaling technology), 3683 (Rabbit monoclonal-HRP against GAPDH, Cell signaling technology), 2G7 (Mouse monoclonal IgG2b against HMGB1 ( )), ab232733 (Mouse monoclonal IgG1 against BioID2, Abcam), 15068 (Rabbit monoclonal-HRP against LaminB1, Cell signaling technology), 4866 (Rabbit polyclonal against VDAC, Cell signaling technology), 7076 (affinity-purified horse anti-mouse IgG-HRP conjugate, Cell signaling technology) and 170-6515 (Goat anti-Rabbit IgG (H + L)-HRP-conjugate, Biorad).

Techniques: Activation Assay, Construct, Titration, Concentration Assay, Expressing, Western Blot, Control

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: The expression of Thrsp is higher in F442A, EB5, and EB7 adipocytes compared to its paralog Mid1ip1 . (A, C) Expression of Thrsp and Mid1ip in preadipocytes and adipocytes, with or without 10 n m T3. The average gene expression ( 2 − Δ C t ) was normalized to the reference gene Rplp0 . (B) Representatives immunoblot of THRSP protein in F442A, EB5, and EB7 cells. A dotted line denotes the location where a lane corresponding to an unrelated treatment was excised from the original blot image. Data represent the mean ± SEM from two independent experiments, each performed in triplicate ( n = 6). Statistical analysis was performed using two‐way ANOVA; * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Expressing, Gene Expression, Western Blot

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Expression of Pparg2 and Thrsp throughout the process of adipose differentiation in EB5 and EB7 cells. Confluent cultures of EB5 or EB7 cells were maintained under nonadipogenic conditions (NAd) or induced to differentiate into adipose cells with RMD, a mix of rosiglitazone, methyl isobutyl xanthine, and dexamethasone, as described in the methodology. (A) Pparg2 gene expression. (B) Thrsp gene expression. Average gene expression ( 2 − Δ C t ) normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Expressing, Gene Expression

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Silencing of Thrsp reduces its expression by 80% in EB5 and EB7 adipocytes, and Mid1ip1 might compensate for Thrsp deletion in EB7 adipocytes. (A, C) Thrsp and Mid1ip1 gene expression levels at 48 and 72 h following knockdown. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05. (B) Representative immunoblot analysis of THRSP in EB5 and EB7 siThrsp adipocytes.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Expressing, Gene Expression, Knockdown, Western Blot

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Effect of Thrsp knockdown on the adipogenic marker Pparg2 , Thrb1 , and Dio2 in brown and brite adipocytes. (A) Pparg2 , (B) Thrb and (C) Dio2 gene expression at 48 and 72 h after knockdown. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Knockdown, Marker, Gene Expression

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Expression of genes related to glucose metabolism and de novo lipogenesis following Thrsp knockdown. (A–D) Expression of Fasn , Gpd1 , Slc2a4 , and Mlxipl in EB7 and EB7 adipocytes. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Expressing, Knockdown, Gene Expression

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Thrsp knockdown increased the expression of genes involved in fatty acid uptake and lipolysis in EB5 and EB7 adipocytes. Expression of (A) Lpl and the lipolytic genes (B) Pnpla2 and (C) Lipe in EB5 and EB7 cells. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Knockdown, Expressing, Gene Expression

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Thrsp knockdown upregulates the expression of genes involved in fatty acid transport in EB5 and EB7 adipocytes. (A, B) Expression of Fabp4 and Cd36 in brown and brite adipocytes was measured at 48 and 72 h after knockdown. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Knockdown, Expressing, Gene Expression

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: The decrease in Thrsp expression downregulated the expression of thermogenic genes in both brown and brite adipocytes. (A–D) Expression of the thermogenic genes Ppargc1a , Cpt1b , Acacb , and Cidea at 48 and 72 h after Thrsp knockdown. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Expressing, Knockdown, Gene Expression

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Effect of silencing Thrsp in the gene expression of Scd1. Thrsp knockdown in EB5 adipocytes caused an increment in Scd1 expression after 48 h in EB5. The average gene expression ( 2 − Δ C t ) was normalized to Rplp0 . The results are the mean ± SEM of two triplicate experiments ( n = 6). Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Gene Expression, Knockdown, Expressing

Journal: Febs Letters

Article Title: Spot‐14 and its paralog Spot‐14 R regulate expression of metabolic and thermogenic pathway genes in murine brown and beige adipocytes

doi: 10.1002/1873-3468.70052

Figure Lengend Snippet: Physiological and molecular responses of Thrsp ‐silenced EB5 and EB7 adipocytes. (A) Representative immunoblot analysis of FABP4. (B) Glycerophosphate dehydrogenase (GPDH) activity. (C) Ucp1 expression in EB5 and EB7 adipocytes stimulated for 24 h with norepinephrine. The results are the mean ± SEM of two or three triplicate experiments to gather at least n = 6. Two‐way ANOVA was used to establish statistical differences indicated with * P ≤ 0.05.

Article Snippet: Proteins were separated using SDS/PAGE, and immunoblotting was done with rabbit polyclonal antibodies against mouse THRSP (1 : 1000) from Protein Tech (Rosemont, IL, USA) and Rab Guanine Nucleotide Dissociation Inhibitor (GDI; Invitrogen), and with POX polyclonal goat anti‐Rabbit 111035003; Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Western Blot, Activity Assay, Expressing