A1 eclipse ti confocal microscope

As described in previous study, whole-cell recordings with voltage clamp at –70 mV for about 10–15 mins. The cesium methanesulfonate internal solution was made as described above with 2 mg/mL Biocytin (Sigma Cat#B4261). Then slices were transferred to 4%PFA/PBS and fixed one hour in room temperature. Slices were washed 3x5 min with PBS, permeabilized in 0.3% Triton-X100/PBS for 30 min, and blocked in 5% normal goat serum (NGS)/0.1% Triton-X100/PBS at room temperature for 1 hr. Subsequently, slices were incubated in Streptavidin Alexa 647 (Invitrogen Cat#S32357) diluted 1:1000 in 5% NGS/0.1% Triton-X100/PBS at 4 °C overnight, washed 5x5 min with PBS and mounted with 0 thickness coverglass (Assistent Cat#01105209). Images were acquired using a Nikon A1 Eclipse Ti confocal microscope with 20 x and 100 x objectives, operated by NIS-Elements AR acquisition software. For spine imaging, Z-stacks were collected at 0.2 μm with 0.06 μm/pixel resolution, and 6–10 dendrites were analyzed per cell.

HEK293T (#CRL-3216) cells were acquired from ATCC, was authenticated and tested negative for mycoplasma contamination. H293T cells were cultured on coverslips coated with poly-L-lysine (Sigma-Aldrich). Cells were transfected with either mRuby, NUP62-mRuby, HA-eGFP, Nup54-HA-eGFP, pEGFP-SF2/SRSF1 (Addgene, #17990), FLAG-NM23-H1/NME1 (Addgene, #25000), Frt-V5-HspB2 (Addgene, #63103), or mRFP-FKBP1A (Addgene, #67514) expression plasmids using TurboFect (Thermo Fisher Scientific), following the manufacturer’s instructions. Cells were washed and incubated with primary antibodies overnight: rabbit anti-TDP-43 (10782–2-AP), 1:1000 (Proteintech); or rat anti-phospho TDP-43 (MABN14), 1:1000 (Millipore, Sigma-Aldrich). Cells were washed and incubated with secondary antibodies: anti-rabbit Alexa Flour 647, 1:100 (Life Technologies, #1660844); anti-rat Alexa Flour 568 (Invitrogen, Cat # A-11077). Coverslips were mounted onto slides using Fluoroshield with DAPI (Sigma-Aldrich, #F6057). Cells were imaged with a NIKON A1 eclipse T_i confocal microscope.