Ab150353

Coverslips with fixed primary neurons were transferred to 4-well slides and recovered in PBS for 10 min. Cells were permeabilized with 0.2% Triton-X100 in PBS for 5 minutes and washed 3 times over 6 minutes with PBS-T (PBS + 0.02% Tween-20; one quick wash followed by 2×3 min). Samples were blocked in PBS-T supplemented with 3% BSA and 2% donkey serum (blocking buffer) for 30 min before incubation with primary antibodies (GFP #A-11122 Invitrogen; RFP #600–401-379 Rockland; Actin #A5441 Sigma; WAVE1 #ab272916 abcam; Cpeb4 #ab224162 abcam; Rbms1 #ab150353 abcam) in PBS-T+3% BSA overnight at 4°C. The next day, coverslips were washed three times in PBS-T (1x fast wash, 2× 5min), incubated with isotype-specific secondary antibodies (Life Technologies) and DAPI (1:10,000, #D1306 ThermoFisher) for 2–3 hours at room temperature in PBS-T+3% BSA on an orbital shaker, followed by three final washes in PBS-T as described above, mounting on a glass slide using fluoromount (#0100–01, VWR/Southern Biotech), and sealing with nail polish. Fluorescence images were acquired using an epifluorescence microscope equipped with a motorized stage and a 40x oil objective (Nikon NiE, EDF z-stack projections through NIS Elements software (Nikon)), as well as a confocal microscope equipped with a 40x oil objective (LSM880, Zeiss, maximum intensity projections of 10 μm z stack).

Western blots were performed using standard tris-glycine SDS-PAGE protocols. Samples had normalized protein concentration assessed using a Bradford assay (#23246, Thermo). Resolved proteins were electroblotted onto PVDF membranes using semi-dry transfer, blocked for 30 min in TBS supplemented with 0.1% Tween-20 and 5% skim milk, and incubated with primary antibodies (GAP43 #MAB347 Millipore; Actb #A5441 Sigma; Gm130 #610823 BD Biosciences; MAP2 #M1406 Sigma; WAVE1 #ab272916 abcam; WAVE2 #3659T Cell Signaling Technologies; WAVE3 #2806S Cell Signaling Technologies; Cpeb4 #ab224162 abcam; Rbms1 #ab150353 abcam; Tau #AF3494 RR&D) in TBS supplemented with 0.1% Tween-20 and 3% BSA over night at 4°C. The next day, the membrane was washed three times in TBS supplemented with 0.1% Tween-20, incubated for 2 hours at room temperature with secondary antibodies that were isotype-specific, HRP-conjugated, and cross-absorbed (Life Technologies), then washed again three times with TBS supplemented with 0.1% Tween-20. Immunoreactive bands were visualized through detection of chemiluminescence using a CCD camera imager (FluoroChemM, Protein Simple). Quantification of signal intensity in the various bands was performed using the standard Fiji software package.