Journal: Cell & Bioscience

Article Title: ANGPTL2 binds MAG to efficiently enhance oligodendrocyte differentiation

doi: 10.1186/s13578-023-00970-3

Figure Lengend Snippet: ANGPTL2-MAG induces Fyn-mediated signaling to enhance the differentiation of oligodendrocytes. A Gene Ontology (GO) analysis of the downregulated differentially expressed genes (DEGs) in the brains of Angptl2 + / + and Angptl2 −/− mice at day 15 as determined by RNA sequencing (n = 3). B Enrichment score plots from GSEA related to the GO signature for myelin sheath and ensheathment of neurons (n = 3). FDR, false discovery rate; NES, normalized enrichment score. C Relative mRNA levels of potential candidates related to myelination markers, transcription factors, metabolic regulators and other genes in the brain tissues of Angptl2 + / + and Angptl2 −/− mice at day 15 as measured by quantitative RT-PCR (n = 3). D Immunoblot analysis of MYRF and ANGPTL2 protein levels in the brain tissues of Angptl2 + / + and Angptl2 −/− mice at day 5, day 15 and day 35. Ratio of MYRF/β-actin was quantified and normalized against Angptl2 + / + , respectively. One representative experiment is shown. E–F Immunoblot analysis of MYRF protein levels in the brain tissues of Mag + / + and Mag −/− mice ( E ) or Mag −/− Angptl2 + / + and Mag −/− Angptl2 −/− mice ( F ) at day 5, day 15 and day 35. Ratio of MYRF/β-actin was quantified and normalized against Angptl2 + / + , respectively. One representative experiment is shown. G – H MAG directly interacted with FYN, as detected by forward ( G ) or reverse ( H ) co-immunoprecipitation assays. CMV-MAG (full-length)-FC and pLVX-FYN-strepII plasmids were used in this experiment. One representative experiment is shown. I RSC96 cells with ectopic expression of MAG (full-length)-FLAG and FYN-StrepII were treated with ANGPTL2 proteins, followed by co-immunoprecipitation analysis to evaluate the changes in tyrosine phosphorylation levels of MAG and FYN using 4G10 and p-SRC (Tyr416) antibodies, respectively. The levels of immunoprecipitated protein were quantified and normalized against the control group, respectively. One representative experiment is shown. J RSC96 cells overexpressing FYN-StrepII or MAG (full-length)-FC were subjected to immunoblot analysis to determine MYRF protein levels. Ratio of MYRF/β-actin was quantified and normalized against negative control (empty vector), respectively. One representative experiment is shown. K Western blot analysis of the protein levels of P-SRC (Tyr416), Fyn and MBP in HCN cells 72 h after induction with IGF1 (100 ng/ml), with/without ANGPTL2-Flag (2 μg/ml) and AZD0530 (2 μM) as indicated. Ratios of P-SRC (Tyr416)/β-actin, Fyn/β-actin, MYRF/β-actin and MBP/β-actin were quantified and normalized against the control treated with IGF1 alone, respectively. One representative experiment is shown. L Schematic diagram of the working model for the role of ANGPTL2-MAG in oligodendrocytes differentiation, myelination and differentiation. (*** p < 0. 001)

Article Snippet: Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies as following: anti-MBP (CST, Cat#78896), anti-MAG (Abcam, ab89780), anti-MOG polyclonal antibody (Santa Cruz Biotech, SC-166172), anti-Fyn (Abcam, ab184276), anti-Phospho-Src family (Tyr416) (CST, Cat #2101), anti-MYRF (Abclonal, A16355), anti-ANGPTL2 (R&D, AF1444) and anti-β-Actin-pAb-HRP-DirecT (MBL, PM053-7).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Expressing, Negative Control, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

doi: 10.1016/j.xcrm.2023.100917

Figure Lengend Snippet:

Article Snippet: Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb , Cell Signaling Technology , Cat# 6943S; RRID: AB_10013641.

Techniques: Purification, Functional Assay, Recombinant, Staining, Clone Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Sequencing, Software

Journal: bioRxiv

Article Title: Active Gαi/o mutants accelerate breast tumor metastasis via the c-Src pathway

doi: 10.1101/2023.01.16.524334

Figure Lengend Snippet: A-B , representative immunoblots showing the expression of Gα i2 R179C and Gα oA R243H in MCF10A cells ( A ) and activation of c-Src and STAT3 by phosphorylation of Src Y416 and STAT3 Y705 in MCF10A cells treated with vehicle control (CT), 2 μM of saracatinib (sara) and STAT3-IN-1 (STAT3i) (B) . CD , the effect of Gα i2 R179C and Gα oA R243H expression on the growth of MCF10A cells in 2D culture ( C ) and in Matrigel ( D ). E , representative images showing the absence of tumor formation in nude mice orthotopically implanted with MCF10A cells expressing GFP, Gα i2 R179C or Gα oA R243H. *, **,*** p<0.05, 0.01 and 0.001 vs. GFP, n=4-6. One-way ANOVA was used for statistical analysis in this Figure.

Article Snippet: Antibodies for EGFR (no. 2232), phospho-EGFR Y1068 (no. 3777), EGFR (no. 4267), HER2 (no. 2165), phospho-HER2 Y1221/1222 (no. 2243), AKT (no. 4685), phospho-AKT S473 (no. 4060), Src (no. 2109), phospho-Src Y416 (no. 6943), ERK1/2 (no. 4696) and phospho-ERK1/2 T202/Y204 (no. 4370) were obtained from Cell Signaling Technology; GADPH (sc-47724) from Santa Cruz Biotechnology; mouse anti-ee antibody (no. 901801) from Biolegend.

Techniques: Western Blot, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Active Gαi/o mutants accelerate breast tumor metastasis via the c-Src pathway

doi: 10.1101/2023.01.16.524334

Figure Lengend Snippet: A-B , Western blotting ( A ) showing increased phosphorylation of c-Src Y416 and EGFR Y1068 and no change in phosphorylation of AKT s473 , STAT3 Y705 and HER2 Y1221/1222 in Neu/Gα i2 R179C tumors as compared to Neu tumors. Each lane represents a sample from an individual tumor. The Western blotting data from A were quantified and expressed as the ratio of the phosphorylated to total proteins ( B ). Two tail unpaired Student’s t test was used for statistical analysis of the data in B , and p values are shown. C-D , Western blotting showing phosphorylation of the indicated proteins in Neu cells expressing GFP, Gα i2 R179C (Gα i2 ) or Gα oA R243H (Gα oA ) and treated with vehicle control (CT) or saracatinib (sara; 2 μM) ( C ) and Neu cells overexpressing GFP or the constitutively active c-Src Y527F mutant ( D) . E , co-immunoprecipitation of ee-tagged Gα i2 R179C and Gα oA R243H with c-Src in Neu cells. Neu cells expressing GFP, Gα i2 R179C (Gα i2 ) or Gα oA R243H (Gα oA ) were immunoprecipitated with the anti-ee antibody and probed with the indicated antibodies.

Article Snippet: Antibodies for EGFR (no. 2232), phospho-EGFR Y1068 (no. 3777), EGFR (no. 4267), HER2 (no. 2165), phospho-HER2 Y1221/1222 (no. 2243), AKT (no. 4685), phospho-AKT S473 (no. 4060), Src (no. 2109), phospho-Src Y416 (no. 6943), ERK1/2 (no. 4696) and phospho-ERK1/2 T202/Y204 (no. 4370) were obtained from Cell Signaling Technology; GADPH (sc-47724) from Santa Cruz Biotechnology; mouse anti-ee antibody (no. 901801) from Biolegend.

Techniques: Western Blot, Expressing, Mutagenesis, Immunoprecipitation