Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Anti-NCAM1 autoantibodies disrupt NCAM1-NCAM1 and NCAM1-GDNF interactions (A) Pull-down assay confirming that IgG purified from a patient with schizophrenia who was positive for anti-NCAM1 autoantibodies disrupts NCAM1-NCAM1 interactions. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose. (B) Pull-down assay showing that IgG purified from a patient with schizophrenia who was anti-NCAM1 autoantibody-positive disrupts the NCAM1-GDNF interaction. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose.

Article Snippet: GDNF Human Recombinant Protein , Origene , Cat# TP760516.

Techniques: Pull Down Assay, Purification

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet:

Article Snippet: GDNF Human Recombinant Protein , Origene , Cat# TP760516.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Software

Journal: The Journal of Experimental Medicine

Article Title: Feeding-induced hepatokine, Manf, ameliorates diet-induced obesity by promoting adipose browning via p38 MAPK pathway

doi: 10.1084/jem.20201203

Figure Lengend Snippet: Manf stimulated browning via p38 MAPK signaling. (A) qPCR analysis of thermogenic genes in primary iWAT incubated with CM from primary hepatocytes infected with Ad-GFP or Ad-Manf for 48 h ( n = 3). (B) Thermogenic gene expression in primary iWAT treated with recombinant Manf and Fgf21 ( n = 3). (C) Phosphorylated of p38 and ATF2 in primary adipocyte from iWAT treated with different concentration of Manf for 30 min. (D) Phosphorylated and total p38 and ATF2 in primary adipocyte from iWAT incubated with 2.5 nM Manf for different periods of time. (E) Primary adipocytes were pretreated with SB203580 (10 µM) for 2 h, then Manf (2.5 nM) for an additional 48 h. mRNA levels of thermogenic genes were detected by qPCR ( n = 3). (F) Phosphorylated and total p38 and ATF2 protein levels in iWAT from WT and Tg mice fed an HFD for 12 wk ( n = 3). Each experiment was independently performed two to three times. All data are mean ± SEM. *, P < 0.05; **, P < 0.01. Ctrl, control.

Article Snippet: The following antibodies were used: rabbit-anti-MANF (1:10,000 for WB; SAB3500384; Sigma-Aldrich), rabbit-anti-Ucp1 (1:4,000 for WB, 1:400 for IHC; Ab10983; Abcam), rat-anti-F4/80 (1:200 for IF; Ab6640; Abcam), mouse-anti-Pgc-1α (1:2,000 for WB; Sc-517380; Santa Cruz Biotechnology), rabbit-anti-p-Akt (1:2,000 for WB; Ser 473; Sc-7985-R; Santa Cruz Biotechnology), rabbit-anti-Akt (1:2,000 for WB; Sc-8312; Santa Cruz Biotechnology), rabbit-anti-HSL (1:2,000 for WB; Sc-25843; Santa Cruz Biotechnology), mouse-anti-p-p38 (1:2,000 for WB; Sc-166182; Santa Cruz Biotechnology), rabbit-anti-p-HSL (1:2,000 for WB; 4126; Cell Signaling Technology), rabbit-anti-ATGL (1:2,000 for WB; 2138s; Cell Signaling Technology), rabbit-anti-p-Plin1 (1:2,000 for WB; 100G7E; Cell Signaling Technology), rabbit-anti-Plin1 (1:2,000 for WB; 9349; Cell Signaling Technology) rabbit-anti-p38 (1:2,000 for WB; BM4142, Boster), rabbit-anti-ATF2 (1:2,000 for WB; BA0653; Boster), rabbit-anti-p-ATF2 (1:2,000 for WB; AP1051; Abclonal), rabbit-anti-β-actin (1:100,000 for WB; AC026; Abclonal), and mouse-anti-β-tubulin (1:4,000 for WB; 200608; Zen Bio Science).

Techniques: Incubation, Infection, Gene Expression, Recombinant, Concentration Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Feeding-induced hepatokine, Manf, ameliorates diet-induced obesity by promoting adipose browning via p38 MAPK pathway

doi: 10.1084/jem.20201203

Figure Lengend Snippet: Manf did not affect cAMP, Erk, and Creb signaling pathway. (A) The expression of Fabp4 in primary adipocytes from iWAT incubated with CM collected from primary hepatocytes infected with Ad-GFP or Ad-Manf ( n = 6). (B) mRNA levels of Fabp4 in primary adipocytes from iWAT treated with recombinant Manf ( n = 3). (C) Phosphorylated and total Erk and Creb in primary iWAT treated with recombinant Manf (2.5 nM; n = 2). (D) The levels of p-p38 in primary adipocytes pretreated with p38 MAPK inhibitor SB023580 (SB; 10 µM) for 2 h followed by Manf (2.5 nM) treatment for 30 min ( n = 2). (E and F) Serum and iWAT norepinephrine (NE) levels in WT and Tg mice on an HFD ( n = 5 or 6). (G) cAMP levels of iWAT from WT and Tg mice on an HFD ( n = 6). (H) Immunofluorescent staining of F4/80 in BAT from HFD-fed mice. (I and J) mRNA levels of markers of M1 and M2 macrophages and inflammatory genes in BAT from HFD-fed mice. Each experiment was independently performed two to three times. All data are mean ± SEM. *, P < 0.05; **, P < 0.01. Ctrl, control.

Article Snippet: The following antibodies were used: rabbit-anti-MANF (1:10,000 for WB; SAB3500384; Sigma-Aldrich), rabbit-anti-Ucp1 (1:4,000 for WB, 1:400 for IHC; Ab10983; Abcam), rat-anti-F4/80 (1:200 for IF; Ab6640; Abcam), mouse-anti-Pgc-1α (1:2,000 for WB; Sc-517380; Santa Cruz Biotechnology), rabbit-anti-p-Akt (1:2,000 for WB; Ser 473; Sc-7985-R; Santa Cruz Biotechnology), rabbit-anti-Akt (1:2,000 for WB; Sc-8312; Santa Cruz Biotechnology), rabbit-anti-HSL (1:2,000 for WB; Sc-25843; Santa Cruz Biotechnology), mouse-anti-p-p38 (1:2,000 for WB; Sc-166182; Santa Cruz Biotechnology), rabbit-anti-p-HSL (1:2,000 for WB; 4126; Cell Signaling Technology), rabbit-anti-ATGL (1:2,000 for WB; 2138s; Cell Signaling Technology), rabbit-anti-p-Plin1 (1:2,000 for WB; 100G7E; Cell Signaling Technology), rabbit-anti-Plin1 (1:2,000 for WB; 9349; Cell Signaling Technology) rabbit-anti-p38 (1:2,000 for WB; BM4142, Boster), rabbit-anti-ATF2 (1:2,000 for WB; BA0653; Boster), rabbit-anti-p-ATF2 (1:2,000 for WB; AP1051; Abclonal), rabbit-anti-β-actin (1:100,000 for WB; AC026; Abclonal), and mouse-anti-β-tubulin (1:4,000 for WB; 200608; Zen Bio Science).

Techniques: Expressing, Incubation, Infection, Recombinant, Staining, Control