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. 2021 Aug 13;12:4926. doi: 10.1038/s41467-021-25227-w

Fig. 6. CENTLEIN directly interacts with SUN5 and PMFBP1.

Fig. 6

a Amino acids 971–1396 of CENTLEIN are necessary to bind to SUN5. HEK293T cells were co-transfected with FLAG-SUN5 and the indicated fragments of MYC-CENTLEIN, immunoprecipitated with anti-MYC antibody, and then immunoblotted with FLAG and MYC antibodies, respectively. +, red, interaction; −, black, no interaction. b Amino acids 601–970 and 971–1396 of CENTLEIN are necessary to bind to PMFBP1. HEK293T cells were co-transfected with GFP-PMFBP1 and the indicated fragments of MYC-CENTLEIN, immunoprecipitated with anti-MYC antibody, and then immunoblotted with MYC and GFP antibodies, respectively. +, red, interaction; −, black, no interaction. c The SUN domain of SUN5 is necessary to bind to CENTLEIN. HEK293T cells were co-transfected with MYC-CENTLEIN and the indicated fragments of GFP-SUN5, immunoprecipitated with anti-GFP antibody, and then immunoblotted with GFP and MYC antibodies, respectively. +, red, interaction; −, black, no interaction. d Amino acids 1–282 and 750–1023 of PMFBP1 are necessary to bind to CENTLEIN. HEK293T cells were co-transfected with MYC-CENTLEIN and the indicated fragments of GFP-PMFBP1, immunoprecipitated with anti-GFP antibody, and then immunoblotted with MYC and GFP antibodies, respectively. +, red, interaction; −, black, no interaction. e The SUN domain of SUN5 directly bind the 971–1396aa region of CENTLEIN. GST-SUN5 129–373aa and GST-SUN5 193–373aa were purified and used to pull down MBP-CENTLEIN 971–1396aa; GST was used as a control. Asterisks in the bottom panel indicate GST products cleaved from the fused proteins. f Amino acids 750–1023 of PMFBP1 directly bind the 601–970aa region of CENTLEIN. GST-PMFBP1 1–282aa and GST-PMFBP1 750–1023aa were purified and used to pull down MBP-CENTLEIN 601–970aa; GST was used as a control. Asterisks in the bottom panel indicate GST products cleaved from the fused proteins. g CENTLEIN acts as a molecular linker between SUN5 and PMFBP1. GFP-PMFBP1, FLAG-SUN5, and empty vector or MYC-CENTLEIN plasmids were co-transfected into HEK293T cells. Twenty-four hours after transfection, cells were collected for immunoprecipitation (IP) with anti-FLAG antibody and analyzed with FLAG, MYC, and GFP antibodies, respectively. The experiment was repeated three times independently with similar results (a–g).