Journal: Cell reports
Article Title: MTHFD2 reprograms macrophage polarization by inhibiting PTEN.
doi: 10.1016/j.celrep.2023.112481
Figure Lengend Snippet: Figure 5. MTHFD2 interacts with PTEN (A and B) Immunoprecipitation of exogenous PTEN-hemagglutinin (HA) with an anti-HA antibody (A) or endogenous MTHFD2 with an anti-MTHFD2 antibody (B) and immunoblotting analyses with the indicated antibodies were performed using HEK293T cell extracts. (C) A GST pull-down assay was performed by mixing bacterially expressed His-PTEN and GST-MTHFD2. (D) Immunoprecipitation of exogenous PTEN-HA with an anti-HA antibody and immunoblotting analyses with the indicated antibodies were performed. PTEN-HA and MTHFD2-FLAG or the FLAG-tagged N-terminal deletion mutant of MTHFD2 were expressed in RAW 264.7 cells. (E) PTEN’s PIP3 phosphatase activity in RAW 264.7 cells transfected with plasmids expressing MTHFD2-FLAG or the FLAG-tagged N-terminal deletion mutant of MTHFD2. The amount of released phosphate was normalized to the amount of immunoprecipitated PTEN determined by the densitometry of western blots. (F) Western blot analysis with the indicated antibodies in WT and MTHFD2-KO BMDMs transduced with MTHFD2-FLAG or the FLAG-tagged N-terminal deletion mutant of MTHFD2. (G and H) Immunoprecipitation of lysates of WT and MTHFD2-KO BMDMs treated with IL-4 (G) or IFN-g (H) for the indicated times and immunoblotting analyses with the indicated antibodies were performed. Data are presented as mean ± SEM. n = 6 per group (E). NS, not significant (p R 0.05), *p < 0.05, **p < 0.01, Student’s t test. See also Figure S7.
Article Snippet: SAKURA Cat #4583 PTEN (aa118-141) peptide: biotinHVAAIHCKAGKGRTGVMICAYLLH Synbio N/A MTHFD2 (aa215-225) peptide: biotin-TDGAHERPGGD Synbio N/A Recombinant His-PTEN protein This paper N/A Recombinant GST protein This paper N/A Recombinant GST-MTHFD2 protein This paper N/A Recombinant GST-MTHFD2-D168E protein This paper N/A Recombinant GST-MTHFD2-R201A protein This paper N/A Critical commercial assays QuikChange II site-directed mutagenesis kit Agilent Technologies Cat #200523 Malachite green phosphate assay kit Sigma-Aldrich Cat #MAK307 Mouse IL-6 ELISA Kit Boster Cat #EK0411 Mouse IL-1b ELISA Kit Boster Cat #EK0394 Mouse CCL22 ELISA Kit Boster Cat #EK0447 Mouse CCL17 ELISA Kit Boster Cat #EK0685 Hematoxylin-eosin stain kit Solarbio Cat #G1120 Sirius red staining kit Solarbio Cat #G1471 Oil red O staining kit Solarbio Cat #G1261 Alanine aminotransferase assay kit Nanjing Jiancheng Bioengineering Institute Cat #C009-2-1 Aspartate aminotransferase assay kit Nanjing Jiancheng Bioengineering Institute Cat #C010-2-1 RNAprep pure micro kit Tiangen Cat #DP420 TransScript II First-Strand cDNA Synthesis SuperMix TransGen Cat #AH301-02 Cell Mito stress test kit Agilent Cat #103015-100 Glycolysis stress test kit Agilent Cat #103020-100 PI3-Kinase activity ELISA kit Echelon Cat #k-1000s Experimental models: Cell lines HEK293T ATCC CRL-3216 HEK293A ATCC CRL-1573
Techniques: Immunoprecipitation, Western Blot, Pull Down Assay, Mutagenesis, Activity Assay, Transfection, Expressing, Transduction
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