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. 2023 Nov 6;7(1):e202302335. doi: 10.26508/lsa.202302335

Figure 4. SYNJ2BP is present in a high-molecular weight complex with ESYT1.

Figure 4.

(A) Characterization of ESYT1 and SYNJ2BP complexes. Heavy-membrane fractions from control human fibroblasts, SYNJ2BP knock-down fibroblasts, fibroblasts overexpressing SYNJ2BP, and fibroblasts overexpressing SYNJ2BP together with a 3XFLAG-tagged version of ESYT1 were analyzed by blue native PAGE. Samples were run in duplicate on the same gel and immunoblotted with anti-SYNJ2BP (left) and anti-ESYT1 antibodies (right). Lower horizontal line: 410 kD complex where both SYNJ2BP and ESYT1 run. Higher horizontal line: higher molecular weight complex observed when SYNJ2BP is overexpressed together with a 3xFLAG-tagged version of ESYT1. (B) Two-dimensional electrophoresis analysis (BN-PAGE/SDS–PAGE) of SYNJ2BP-interacting proteins in control human fibroblasts and fibroblasts overexpressing SYNJ2BP. The migration of known protein complexes in the first dimension is indicated on the top of the blot (UQCRC1: OXPHOS complex III at 500 kD, NDUFA9: OXPHOS complex I at 1,000 kD). The position of identified SYNJ2BP containing complexes and their alignment with ESYT1 and RRBP1 containing complexes are indicated with grey lines. (C) Characterization of ESYT1, SYNJ2BP, and RRBP1 complexes. Heavy-membrane fractions from fibroblasts overexpressing SYNJ2BP or fibroblasts overexpressing SYNJ2BP in which either ESYT1 or RRBP1 was knocked down were analyzed by Blue-Native PAGE. Samples were run in triplicate on the same gel and immunoblotted with anti-ESYT1 (left), anti-SYNJ2BP (center), and anti-RRBP1 antibodies (right). (D) RRBP1, ESYT1 and SYNJ2BP protein levels in fibroblasts overexpressing SYNJ2BP untreated or treated with puromycin (200 μM for 2h and 30 mins). Whole-cell lysates were analyzed by SDS–PAGE and immunoblotting. CCDC47 was used as a loading control. (E) Two-dimensional electrophoresis analysis (BN-PAGE/SDS–PAGE) of SYNJ2BP-interacting proteins in fibroblasts overexpressing SYNJ2BP untreated or treated with puromycin (200 μM for 2h and 30 mins). The position of identified SYNJ2BP-containing complexes and their alignment with ESYT1 and RRBP1-containing complexes are indicated with grey lines.