Mms 118p

The following antibodies were used: mouse Tau-1 (Chemicon, MAB3420, 1:200), mouse anti-MAP2 (SigmaAldrich, M4403, 1:1500), rabbit anti-MAP2 (Abcam, ab32454, 1:1000), mouse anti-Ankyrin-G (Antibodies Inc., 75–146, 1:100), rabbit anti-Plexin-A1 (Abcam, ab23391, 1:1000), mouse anti-GFP (Covance, MMS-118P, 1:1000), mouse anti-FLAG M2 (SigmaAldrich, F3165, 1:1000), anti-Rap1 (Upstate, #07–916, 1:200), anti-Sema3A (Abcam, ab23393, 1:200), SMI-312 (BioLegend, 837904, 1:200) and goat secondary antibodies labeled with Alexa-350 (Molecular Probes, 1:200), −488 (1:800) or −594 (1:800). Nuclei were stained with Hoechst 33342 (Molecular Probes, 1:6000).

Cells were lysed with ice-cold RIPA buffer (Boston BioProducts) in the presence of 1 × protease inhibitor cocktail (Sigma), 1 mM NaF and 1 mM Na₃VO₄. Total cell lysates were mixed with 2 × SDS-loading buffer and were boiled at 95 °C for 10 min. Protein samples (10–100 μg) were then loaded and separated on 4–12% gradient SDS–PAGE gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 1% bovine serum albumin in PBS supplemented with 0.1% Tween 20 and were incubated with antibodies against GFP (1:10,000; Covance, MMS-118P), LC3 (1:2,000; Sigma, L8918), Lamin A/C (1:1,000; Cell Signaling Technology, 4777), Lamp1 (1:1,000; Developmental Studies Hybridoma Bank, H4A3), γ-tubulin (1:4,000; Sigma, T5326), TFEB (1:1,000; Cell Signaling Technology, 4240) and TFE3 (1:4,000; Sigma, HPA023881), respectively. Bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit (65-6120) or anti-mouse (62–6520) secondary antibodies (1:5,000, Invitrogen) and enhanced chemiluminescence reagent (Amersham Pharmacia Biotech). Band intensities were quantified in Image J software. Full versions of all blots are shown in Supplementary Fig. 36.