Nature research paper: Phase separation of FSP1 promotes ferroptosis
BL21 cells were transformed with constructs encoding hsNMT1 and FSP1 . Purification was performed as for wild-type FSP1 with a final step of gel filtration chromatography. Purified protein was obtained and confirmed by denaturing SDS gel. Confirmation of the presence of myristoylated protein was obtained with MS.Myristoylation was confirmed by LC–ESI–MS . Proteins were separated on an Acquity UPLC Protein BEH C4 column , and data were analysed using Masslynx v.4.2 .
To measure turbidity, different concentrations of non-myr-FSP1 and PEG were reconstituted in 10 µl in a 384-well plate and the absorbance at 600 nm was measured using a SpectraMax iD5 microplate reader . To show non-myr-FSP1 condensation in PCR tubes, images were acquired using a smartphone. Representative bright-field images of non-myr-FSP1 condensates on an objective slide were captured using an Eclipse Ts2 microscope with a ×40 objective.
For sedimentation assays, recombinant non-myr-FSP1 was mixed with the same amount of TBS with 0% or 20% PEG and 1 mM DTT and samples were centrifuged at 2,500for 5 min. The supernatant was collected in a new tube and the pellet was resuspended in TBS supplemented with 1 mM DTT. Supernatant and resuspended non-myr-FSP1 were collected by adding 6× SDS sample buffer and subsequently resolved by SDS–PAGE. One gel was subjected to western blotting and probed with anti-FSP1 .
To quantify the FRAP rate, a region of interest for each condensate in the photobleached area and one condensate in a non-photobleached area was determined using ImageJ/Fiji and the mean fluorescence intensity of condensate
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