, Proteins were transferred to Nitrocellulose membrane and incubated with mouse monoclonal anti-GFP (Roche #11814460001) or RFP (ThermoFisher Scientific, MA5-15257) antibody at 1:2000 dilution for 12 h at 4°C. The membranes were then incubated with HRP conjugated secondary anti mouse antibody, Cell signaling 7076S) at 1:2000 dilution. Blot pictures were taken by Bio-rad ChemiDoc XRS + imaging system

. Gst-pulldown and . Assay, GST-pull down assay was modified from previously published protocol 78 . To obtain GST-fusion proteins

E. Arcticexpress and . Coli-strain-(agilent, USA) transfected with pGEX-3X vectors were further cultured in 100 mL LB medium with 100 mg/mL ampicillin, grown for 2 h at 37°C with shaking until OD600 reached 0.6-0.8. Protein expression was induced with 1 mM IPTG and shaking at 11°C for 24 h. The culture was centrifuged at 5000 × g for 10 min at 4°C, the pellet was re-suspended in 10 mL ice-cold bacto-lysis buffer

, The lysed samples were then centrifuged at 15,000 × g for 30 min at 4°C. Supernatants were incubated with glutathione-Sepharose 4B beads (GE17-0756-01, Sigma) at 4°C for 12 h. Coding sequences of FRM-3B, FERM + FA, FERM, LIN-2B or UNC-49B_loops were cloned into pcDNA3.1(+) vector with a HA epitope tag to the N-terminus for in vitro expression. HA tagged proteins were transcribed and translated using TnT® Quick Coupled system, then incubated with GST-fusion coated beads for 12 h at 4°C. The beads were then washed five times with 10 mM STE

H. Biorad and C. A. , After blocking by 5% non-fatty milk, the blot was probed with anti-HA antibodies (#3724, Cell signaling tech.) at 1:1000 dilution, anti-GST (#2624, Cell signaling tech.) at 1:2000 dilution or stained immediately by ponceau S red. Immunohistochemical staining. The N2, frm-3(gk585) or lin-2(n397) animals were grown and collected from NGM rich medium plates. A freeze/cracking step was performed before acetone/methanol fixation at ?20°C. Primary antibody rabbit anti-UNC-49 was diluted at 1:250; mouse anti-UNC-17 (VAChT) was diluted at 1:500 79, Tween-20) buffer. Beads were boiled 10 min after the addition of an equal volume of 2×SDS sample buffer

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, Correspondence and requests for materials should be addressed to B.P.-L. or J.-L.B. Peer review information Nature Communications thanks Erik Lundquist, Katharine Smith and the other, anonymous, reviewer(s) for their contribution to the peer review of this work