Figure 2. Identification of MLF2 as a... | Rockefeller University Press

Figure 2.

$Identification of MLF2 as a molecular component of nuclear herniations in Torsin-deficient cells.(A) Overview of the subcellular fractionation and K48-Ub immunoprecipitation (IP) workflow. K48-Ub–linked candidate proteins were selected based on their fold enrichment in 4TorKO cells over WT cells, and percent sequence coverage. Electron micrograph of NEs isolated from 4TorKO cells with immunogold labeling for K48-Ub is shown. Black arrow, NPC; white arrow, NE herniation containing K48-Ub. (B) Confocal microscopy of 4TorKO cells transfected with MLF2-HA (red), Flag-Emerin (red), or DNAJB2-HA (red) and counterstained with anti–K48-Ub (green) to label NE blebs. Cells stained with anti-VCP (red) serve as a negative control for the selection criteria. Scale bar, 10 µm; inset, 2 µm. (C) Western blot analysis of the subcellular fractions isolated from the workflow presented in A using WT and 4TorKO cells stably expressing MLF2 with an endogenous C-terminal 3xHA tag. SUN2 was used as a NE marker. (D) Validation of the MLF2 association with K48-Ub chains in 4TorKO cells by coIP. KO, Torsin-deficient cells; N, nuclear fraction; NE, NE fraction; and NP, nucleoplasmic fraction.$

Identification of MLF2 as a molecular component of nuclear herniations in Torsin-deficient cells. (A) Overview of the subcellular fractionation and K48-Ub immunoprecipitation (IP) workflow. K48-Ub–linked candidate proteins were selected based on their fold enrichment in 4TorKO cells over WT cells, and percent sequence coverage. Electron micrograph of NEs isolated from 4TorKO cells with immunogold labeling for K48-Ub is shown. Black arrow, NPC; white arrow, NE herniation containing K48-Ub. (B) Confocal microscopy of 4TorKO cells transfected with MLF2-HA (red), Flag-Emerin (red), or DNAJB2-HA (red) and counterstained with anti–K48-Ub (green) to label NE blebs. Cells stained with anti-VCP (red) serve as a negative control for the selection criteria. Scale bar, 10 µm; inset, 2 µm. (C) Western blot analysis of the subcellular fractions isolated from the workflow presented in A using WT and 4TorKO cells stably expressing MLF2 with an endogenous C-terminal 3xHA tag. SUN2 was used as a NE marker. (D) Validation of the MLF2 association with K48-Ub chains in 4TorKO cells by coIP. KO, Torsin-deficient cells; N, nuclear fraction; NE, NE fraction; and NP, nucleoplasmic fraction.