Journal: Cell reports

Article Title: NEIL3 contributes to the Fanconi anemia/BRCA pathway by promoting the downstream double-strand break repair step.

doi: 10.1016/j.celrep.2022.111600

Figure Lengend Snippet: Figure 5. NEIL3 physically binds the CtIP-MRN-DNA2 complex (A) Western blots to show the indicated proteins in subcellular fractions of WT and NEIL3/ HeLa cells after treatment with or without 200 nM cisplatin for 24 h. WCL, whole cell lysate. (B) The levels of the indicated protein in chromatin were normalized to H2AX and then compared with the untreated WT HeLa cells. Data presented are mean ± SEM from three independent experiments. ns, not significant, **p < 0.01, ***p < 0.001, two-tailed Student’s t test. (C) The transcriptional level of CtIP was determined by qPCR in WT and NEIL3/ HeLa cells. (D and E) CtIP level was determined in NEIL3/ HeLa cells treated with 20 mM MG-132 for 2 h (E) CtIP level was determined in NEIL3/ HeLa cells transfected with 0.5mg HA-NEIL3 plasmid. (F) Immunoprecipitation with exogenously expressed NEIL3 to detect its interaction with CtIP, MRN, and DNA2 proteins. Immunoprecipitation was performed using anti-HA magnetic beads. (G) Immunoprecipitation of endogenous NEIL3 to detect its interaction with CtIP, MRN, and DNA2 proteins in HeLa cells synchronized in S-phase. (H) Identify key domain of NEIL3 that binds to CtIP using the mutant constructs. Immunoprecipitation was performed using an anti-GFP antibody. Blots were detected by indicated antibodies.

Article Snippet: IP was then performed by using the Immunoprecipitation (IP/CoIP) Kit (Absin) with the NEIL3 antibody (Proteintech) and normal rabbit IgG (Cell Signaling Technology), respectively.

Techniques: Western Blot, Two Tailed Test, Transfection, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Mutagenesis, Construct