Human Flag-tagged RIG-I IP-MS in HEK293T

ABSTRACT: nevaeh: The PARP inhibitor olaparib is widely used in cancer but is underused in treating lung adenocarcinoma (LUAD). Our initial analysis revealed that RIG-I, which is typically found in the cytoplasm and activates innate immunity, is linked to LUAD outcome. Here, we discovered that RIG-I undergoes lactylation, with the acetyltransferase PCAF serving as the lactylation writer. Treatment with the lactate transporter inhibitor syrosingopine prevents the efflux of lactic acid from cancer cells, increasing intracellular lactic acid, promoting RIG-I lactylation, and enhancing its interaction with the nuclear transport protein importin 8. The augmented interaction facilitates the nuclear translocation of RIG-I, diminishing PARP1 activity and enhancing the therapeutic effects when combined with olaparib. This synergy disrupts PARP1-mediated DNA repair mechanisms, increasing drug sensitivity and inhibiting tumor growth. The combination of olaparib and syrosingopine demonstrates better therapeutic efficacy than olaparib monotherapy in LUAD, providing a promising strategy for future LUAD treatments. nevaeh: HEK293T cells were transfected with Flag-RIG-I for 48 h, lysed in NP-40 buffer, and sonicated using a JY92-II sonicator. The supernatant was first incubated overnight with an anti-Flag conjugated with beads (Sigma, A2220) after being denatured. Following centrifugation at 3,000 rpm (4°C for 5 min), the supernatants were completely removed, and 60 μL of Flag–peptide was added to competitively bind to the beads. RIG-I protein was released, precipitated using Trichloroacetic acid (TCA), and subjected to mass spectrometry analysis for lactylation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Lung Adenocarcinoma

SUBMITTER: yulin li  

LAB HEAD: Yulin Li

PROVIDER: PXD058597 | Pride | 2025-12-10

REPOSITORIES: Pride