Nup133 is required for nuclear pore basket assembly and dynamics in embryonic stem cells. B. Souquet et al.
Pull-down experiments using as bait Nup153-NTD[aa1-245](negative control) or [aa1-339] and whole cell extracts from WT (HM1) or Nup133-/- (KO, #14) mESCs. Inputs (1x equivalent) and eluates (64x equivalent, dataset (i) or 48x equivalent, dataset (ii)) were analyzed by western blot. These western blots revealed a similar enrichment of Tpr and Y-complex proteins (Nup107,Nup96, Nup85) in Nup153-NTD [aa 1-339]-purified extracts from either WT or Nup133-/- mESCs. Anti-Nup98 and GAPDH were used as negative controls. The zip-file includes 2 folders correspond to datasets (i): Souquet et al Figure 4D (i, 64x eluates) and (ii): Souquet et al Figure 4D (ii, 48x eluates), respectively. In the dataset (ii), 3 independently processed eluates were analyzed. Each folder encompasses a general montage, in which the areas used for Figure 4C are indicated in red, as well as 5 sub-folders corresponding to the original 16-bit images (each include the ECL signal and the image of the membrane to visualize the molecular markers). For each pull-down experiment, the same samples were loaded on both nitrocellulose (that was stained with Ponceau) and PVDF membranes. - The top of the nitrocellulose membrane was first probed with Nup107, cut, and reprobed with anti-Nup133 His-Nup153 pull-down (nitro I or ii) Nup107 His-Nup153 pull-down (nitro I or ii) Nup133 (rehybr after Nup107) - The bottom of the nitrocellulose membrane was probed with anti-GAPDH antibody His-Nup153 pull-down (nitro i) GAPDH - The PVDF membrane was first cut and probed with Tpr, Nup96 and Nup85 His-Nup153 pull-down (PVDF i)Tpr-Nup96-Nup85 - The part of the PVDF membrane probed wiuth Nup98 was then reprobed with anti Nup98 His-Nup153 pull-down (PVDF i)_Nup98 (rehyb from Nup96)