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, 100-300 nm at low frequency (1-2 kHz). PFT mode decreases the lateral and shear forces, Images were, 2048.
, and 20 mM MgCl 2 was incubated for 1 h at 37 C. The enzyme was denatured at 65 C for 10 min and precipitated proteins were removed by centrifugation. An in vitro poly(ADP-ribosyl)ation assay was performed in the reaction mixtures (20 mL) contained 20 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM DTT, 5 mM MgCl 2 , 400 mM Urea, 0.05 A260/mL of DNase I-activated calf thymus DNA, 50 nM PARP-1, 0.3 mM NAD + , 0.4 mCi [ 32 P]-NAD + and 2 mg FUS, FUSDLCD, G3BP1, HuR, TDP-43, or BSA as indicated at the figure legends. The reactions were initiated by the addition of NAD + . The reaction mixtures were incubated at 37 for 30 min and stopped by adding SDS-sample buffer and heating for 5 min at 90 C. The reaction mixtures were analyzed by 10% SDS-PAGE with subsequent phosphorimaging and/or colloidal Coomassie staining. For the analysis of the degradation of PARP-1-bound PAR by PARG, 30 nM PARP-1 was incubated with 3 nM damaged pBR in the buffer containing 12.5 mM HEPES-KOH, pH 8.0, 25 mM NaCl, 1 mM DTT, 5 mM MgCl 2 , 260 mM Urea, 400 mM, 0.3 mM NAD + , 0.4 mCi [ 32 P]-NAD + at 37 C for 30 min followed by addition of EDTA to the final concentration of 15 mM. After that, the samples were supplemented with 2.4 mM FUS, FUSDLCD, G3BP1, HuR or TDP-43 and incubated at 37 C for 5 min, and then PARG was added to final concentration 4 nM followed by incubation for 1-5 min at 37 C. The reaction mixture, NAD + labeled on the adenylate phosphate was synthesized in a reaction mixture (100mL) containing 2 mM b-Nicotinamide mononucleotide, 1 mM ATP and 0.25 mCi of
, After that the reactions were incubated with RNA-binding proteins (1 mM) for 5 min at 37 C. For sedimentation analysis, the reactions were centrifuged at 10,000 3 g for 5 min at 25 C. Supernatants were collected, and pellets were resuspended in 20 mL of the buffer with 0.25 volume of the loading solution containing 5% SDS, 5% 2-mercaptoethanol, 0.3 M Tris-HCl (pH 6.8), 50% glycerol, and 0.015% bromophenol blue. Supernatants and pellets were divided into two parts. One part (20 mL) was analyzed by separation in 10% SDS-PAGE and visualized with Coomassie staining. The other part (20 mL) was resolved in a 0.8% agarose gel in 0.5 3 TAE buffer at room temperature at 5 V/cm and stained with GelRed. Immunofluorescence analysis Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 45 min at 37 C. After washing with PBS, coverslips were kept with blocking buffer (50 mM Tris pH 7.5, 100 mM NaCl, BSA 2%, 0.15% Triton X-100) for 40 min at 37 C in order to permeabilize the cells and reduce nonspecific recognition by antibodies. Blocking buffer was removed and cells were washed and then incubated for 1 h at room temperature with primary antibody described in supplementary methods. The cells were washed 5 times with PBS and incubated for 1 h with fluorochrome (Alexa Fluorâ488 and À594)-coupled secondary antibodies in 50 mM Tris pH 7.5. After final washes with PBS, the cells were stained with 300 nM DAPI to visualize the nuclei and mounted for fluorescence microscopy analysis. Western blot analysis of PARG expression Cells were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mm EDTA, and protease inhibitor mixture. Lysates were centrifuged at 14,000 3 g for 15 min at 4 C, and supernatants were collected. Proteins (120 mg) of cell extracts were separated on 12% SDS-PAGE gels and transferred onto a PVDF membrane. The membranes were blocked in 5% (w/v) non fat dried milk, C, and the products were separated by denaturing electrophoresis in 20% polyacrylamide gel followed by visualization with phosphorimaging. Analysis of binding of FUS to RNA by EMSA To assay stability of RNA-protein complexes in the presence of PAR, the reactions were performed in a mixture 25 mL containing 12.5 mM HEPES, pH 8.0, 25 mM KCl, 260 mM urea, 0.8 nM mRNA, 30-120 nM FUS and 0.4-4 mM PAR, as indicated in the figure legends