Bovine serum albumin fraction 5

Each aliquot of M. brevicollis cells was incubated in priming buffer (40 mM HEPES-KOH, pH 7.5; 55 mM lithium citrate; 50 mM l-cysteine; 10 % [wt/vol] PEG 8000; and 2 µM papain) to remove the extracellular material coating the cell. The 100 µl aliquots, which contained 5 × 10⁶ cells, were centrifuged for 5 min at 1700× g. The supernatant was removed, and cells were resuspended in 100 µl of priming buffer and then incubated for 35 min at room temperature. Priming was quenched by adding 4 µl of 50 mg/ml bovine serum albumin-fraction V (Thermo Fisher Scientific, Waltham, MA; Cat. No. BP1600-100) and then centrifuged for 5 min at 1250× g and 22 °C with the centrifuge brake set to a “soft” setting. The supernatant was removed with a fine-tip micropipette, and the cells were resuspended in 25 µl of SG Buffer (Lonza).

Each aliquot of M. brevicollis cells was incubated in priming buffer (40 mM HEPES-KOH, pH 7.5; 50 mM lithium citrate; 50 mM l-cysteine; 15 % [wt/vol] PEG 8000; and 2 µM papain) to remove the extracellular material coating the cell. The 100 µl aliquots, which contained 5 × 10⁶ cells, were centrifuged for 5 min at 1700× g and at room temperature. The supernatant was removed, and cells were resuspended in 100 µl of priming buffer and then incubated for 35 min. Priming was quenched by adding 10 µl of 50 mg/ml bovine serum albumin-fraction V (Thermo Fisher Scientific, Waltham, MA; Cat. No. BP1600-100). Cells were then centrifuged for 5 min at 1250× g and 22 °C with the centrifuge brake set to a ‘soft’ setting. The supernatant was removed with a fine-tip micropipette, and the cells were resuspended in 25 µl of SG Buffer (Lonza).

Separate 96 well plates (Greiner Bio-one) were coated with calf thymus DNA (R&D) at 10 μg/mL and incubated overnight at 4°C. Plates were washed with D-PBS (Thermo Scientific) then blocked with D-PBS containing 2% bovine serum albumin fraction V (Thermo Scientific) for 1 h at room temperature (RT). After washing with D-PBS containing 0.05% Tween-20 (Sigma), serum samples were serially diluted in D-PBS containing 2% bovine serum albumin fraction V and 5 μg/mL Heteroblock (Omega Biologicals) then added to the plates and incubated for 2 h at RT. After washing with D-PBS containing 0.05% Tween-20, 1:2,500 dilutions of goat anti-mouse H+L chain HRP (Jackson Immunoresearch), goat anti-mouse IgM (μ chain) HRP (Jackson Immunoresearch) or goat anti-mouse IgG (Fcγ) HRP (Jackson Immunoresearch) were added to the plates and incubated for 90 min at RT. Plates were washed with D-PBS containing 0.05% Tween-20 then developed for 5 min with SureBlue Reserve TMB substrate (SeraCare). Reactions were stopped using 1 N HCL (Thermo Scientific) and plates were read at 450λ using a Molecular Devices Spectra Max 340 PC running Soft Max Pro 3.1.2.

Cells were plated over sterilized coverslips in 6-well plates and transfected as above using mCherry-FUS, pEGFP-LMNA and myc- or FLAG-tagged SETX constructs. Constructs were expressed for 72 h in culture medium as described above. Coverslips were then washed with 1 × PBS and fixed for 15 min in 4% formaldehyde in PBS at RT, washed gently 3 times with 1 × PBS, and permeabilized with 2% Triton-X-100 in 1 × PBS for 10 min. Blocking was performed with 10% bovine serum albumin Fraction V (ThermoFisher) for 1 h at room temperature. Primary antibodies (as needed to detect SETX and endogenous proteins; see Supplemental Table 1 for all antibodies used in these studies) were then added and incubated for 1 h at RT. Cells were washed in PBS twice for 10 min each and then incubated with secondary antibody (see Supplemental Table 1) for 1 h. Cells were washed again in PBS then incubated with Hoechst for 5 min, washed twice in PBS for 5 min. For cells that did not require antibodies for detection of expressed proteins from transfected constructs, cells were fixed, washed in PBS, stained with Hoechst without permeabilization or incubation steps. Coverslips were then inverted and mounted to glass slides using Immu-Mount aqueous mounting medium (ThermoFisher) and imaged using a SP8 Leica confocal microscope.