Fig. 2: SIRT1 is physically associated with the CRL4B complex.

A Immunoaffinity purification and mass spectrometry analysis of SIRT1-containing protein complexes. Whole-cell extracts from PANC-1 cells stably expressing FLAG (Vector) or FLAG-SIRT1 were immunopurified using anti-FLAG affinity columns and eluents with FLAG peptide. Eluates were resolved using SDS-PAGE and silver-stained. Protein bands were retrieved and analyzed using mass spectrometry. B Western blot analysis of the purified fractions using antibodies against the indicated proteins. C Co-IP assays in PANC-1, AsPC-1, BxPC-3, and MIA PaCa-2 cells with anti-SIRT1, followed by IB with antibodies against the indicated proteins, or with antibodies against the indicated proteins followed by IB with anti-SIRT1. D SIRT1 and CRL4B complex co-fractionation using fast protein liquid chromatography. PANC-1 cell nuclear extracts were first fractionated on a DEAE sepharose column, and then on a Superose 6 gel filtration column. Fractions were analyzed using western blotting. Molecular weight standards (kDa) are shown at the top. E GST pull-down assays with bacterially expressed GST-fused proteins and in vitro-transcribed/translated proteins. F Identification of the essential domains required for interaction. (a) GST pull-down assays with GST-fused SIRT1 N-terminal domain (N), NAD⁺- dependent deacetylase catalytic core domain (M), or amino C-terminal domain (C) and in vitro-transcribed/translated CUL4B or DDB1. (b) GST pull-down assays with GST-fused CUL4B DDB1-interacting domain (DID), Cullin domain (Cullin), or NEDD8 neddylation domain (NEDD8) and in vitro-transcribed/translated SIRT1. (c) GST pull-down assays with GST-fused CUL4B NEDD8 neddylation domain (NEDD8) or NEDD8 domain with neddylation site deletion (∆Neddylation) and in vitro-transcribed/translated SIRT1. ∆N, ∆Neddylation. (d) GST pull-down assays with three GST-fused DDB1 propeller domains (BPA, BPB, or BPC) and in vitro-transcribed/translated SIRT1. G Schematic diagram depicting molecular interactions between SIRT1 and CRL4B. H Western blotting analysis of H3K9ac, H3K14ac, H4K16ac, and H2AK119ub1 global levels in PANC-1 cells upon FLAG-tagged SIRT1 or CUL4B overexpression or knockdown. Histone H3, H4, or H2A served as the loading control. Quantitative protein expression by gray scanning. S1, SIRT1; C4, CUL4B. Error bars represent the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01; two-tailed unpaired t test.