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Identification of E3 Ubiquitin Ligase proteins with FOXJ1 and M2 Protein by Co-IP combined with mass spectrometry

ABSTRACT: This project aims to systematically identify E3 ubiquitin ligases interacting with transcription factor FOXJ1 and M2 protein. Three experimental strategies were employed: (1) Co-transfection of FOXJ1-Flag and M2-myc in HEK293 cells, immunoprecipitated with IgG and myc antibodies; (2) Co-transfection of FOXJ1-Flag and M2-myc in HEK293T cells, immunoprecipitated with Flag antibody; (3) Overexpression of M2-myc in primary epithelial cells cultured under air-liquid interface differentiation, immunoprecipitated with IgG and myc antibodies.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lei Wang

PROVIDER: PXD081346 | iProX | Sun Jul 19 00:00:00 GMT+01:00 2026

REPOSITORIES: iProX

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Project description:Tumor-associated macrophages (TAMs), often adopting an immunosuppressive M2-like phenotype, correlate with unfavorable cancer outcomes. Our investigation unveiled elevated expression of the butyrophilin (BTN)2A1 in M2-like TAMs across diverse cancer types. We developed anti-BTN2A1 monoclonal antibodies (mAb) and notably, one clone demonstrated a robust inhibitory effect on M2-like macrophage differentiation, inducing a shift towards an M1-like phenotype both in vitro and ex vivo in TAMs from patients with cancer. Macrophages treated with this anti-BTN2A1 mAb exhibited enhanced support for T-cell proliferation and IFNγ secretion. Indeed, M1-like and M2-like macrophage differentiation involves complex signaling pathways leading to specific gene expression profiles. Typically, stimuli from the TME induce pathways including JAK/STATs, MAPK, PI3K/AKT, NOTCH and NF-κB influencing TAMs polarization. We investigated the signaling pathways activated in monocytes and M-CSF-induced M2-like macrophages upon BTN2A1 engagement using anti-BTN2A mAb5. After 30 min of treatment, we observed phosphorylation of MAPKs (i.e. ERK1/2, P38 and JNK) in CD14+ monocytes and in M2-like macrophages, unlike isotype control-treated cells. In contrast, the PI3K/AKT, JAK/STAT, and NF-kB pathways were not consistently activated after 30 min of anti-BTN2A mAb5 treatment. Since BTN2A1 lacks intrinsic kinase activity, we explored whether BTN2A1 could form complexes with molecular partners in M2-like macrophages that provide kinase activity. We prepared lysates from M2-like macrophages treated with mAb5 or with its isotype control for 30 min and performed immunoprecipitation (IP) using another anti-BTN2A mAb followed by western blotting with anti-phosphoTyrosine (pTyr). A 70 kDa was co-immunoprecipitated with BTN2A1 and revealed by the anti-pTyr in lysates from anti-BTN2A mAb5-treated M2-like macrophages. To identify which Tyrosine kinase is enriched in anti-BTN2A mAb5-treated M2-like macrophages, we performed LC-MS/MS on anti-pTyr immunoprecipitates from treated cells. Several tyrosine kinases, including P38α/δ and JNK, were enriched. Notably, SYK was also identified and matched the 72kDa molecular weight of the pTyr band co-immunoprecipitated with BTN2A1.

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Identification of E3 Ubiquitin Ligase proteins with FOXJ1 and M2 Protein by Co-IP combined with mass spectrometry

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