Rat antihemagglutinin ha

Mouse ES cells were provided by the Crick Institute Biological Resource Unit and were derived from hybrid blastocysts generated by the mating of C57BL/6J and 129 (S6)SvEv mice as previously described (29 (link)).
Unlabeled l-arginine and l-lysine, (R0K0, Sigma-Aldrich, Gillingham, United Kingdom), medium l-arginine [¹³C₆] and l-lysine [D₄] R6K4) and heavy l-arginine [¹³C_6,¹⁵N₄] and l-lysine [¹³C_6,¹⁵N₂] (R10K8) in their hydrochloride forms were obtained from CK Isotopes (Ibstock, United Kingdom).
Rat antihemagglutinin (HA; Roche, Burgess Hill, West Sussex, United Kingdom; 1:1000), rabbit anti-Arl8b (Biorbyt, Cambridge, United Kingdom; 1:200), goat anti-LIMP2 and anti-CAR (R&D Systems, Abington, United Kingdom; both 1:100) primary antibodies were used for immunofluorescence. For western blots, we used the following: mouse anti-Rab5 (Synaptic Systems, Göttingen, Germany; 1:500), rabbit anti-Rab7 (Cell Signaling Technology, Leiden, The Netherlands; 1:1000), rabbit anti-Arl8b (1:500) (30 (link)), goat anti-CAR (R&D Systems, 1:500) and mouse anti-βIII-tubulin (Covance, London, United Kingdom; 1:1000).

Primary cortical neurons and Neuro-2a cells were fixed and permeabilized (12 (link)). Primary neuron cultures were incubated with primary antibody overnight at 4°C with 1:1,000 rabbit anti-neuron-specific nuclear protein (NeuN) (EMD Millipore), 1:12,000 mouse anti-FLAG (Sigma-Aldrich), or 1:2,000 guinea pig anti-synaptophysin. Neuro-2a cells were also incubated with 1:2,000 rat anti-hemagglutinin (HA; Roche) or with 1:2,000 rabbit anti-synaptic vesicle glycoprotein 2C (SV2C) (Synaptic Systems) or in buffer without primary antibody (12 (link)). For Neuro-2a cells, coverslips were mounted and cured in ProLong Gold (Life Technologies) on glass slides. For neurons, Citifluor AF-3 antifade reagent (Electron Microscopy Sciences) was added prior to analysis. Micrographs were collected using a Nikon inverted microscope by epifluorescence using a CFI Plan Apo 60× oil objective (numeric aperture, 1.49) and 20× objective (numeric aperture, 0.45) and a Photometrics CoolSnap HQ2 camera. For primary neuron translocation quantitation, micrographs were collected at 20×. For primary neuron binding and Neuro-2a entry, respectively, 60× micrographs and Z-stacks were taken (12 (link)).