Novex gels

Indicated samples were loaded on 4% to 20% Novex gels (Cat# EC60285BOX; Thermo Fisher Scientific). Proteins were transferred for 1 hour at 120 V using TGS (25 mM Tris pH = 8.5, 192 mM glycine, 0.1% (mass/vol) SDS), 20% (vol/vol) methanol as transfer buffer to polyvinylidene difluoride membranes 0.45 µm (Cat# IPVH00010; Millipore), preactivated in pure methanol. After transfer, the membranes were blocked at RT for 1 hour with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), 5% (mass/vol) nonfat dry milk, then incubated separately in indicated primary antibodies in TBST, 5% (mass/vol) BSA, overnight at 4˚C. Following incubation in horseradish peroxidase-conjugated secondary antibodies from Jackson immunoresearch, blots were revealed by enhanced luminescence (WBKLS0500; Millipore) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software by Silk Scientific Inc.

The nanodisc-laden synaptosomal membrane proteins were incubated with vehicle (0.01% DMSO) or 10 µM t-CNRP1 for 15 minutes on ice and then subjected to immunoprecipitation with isotype-specific IgG or 5 µg CRMP2 antibody, overnight at 4˚C, as described previously.^{56 (link)} After elution in 25 mM glycine (pH 2.0), the sample was brought up to 1.0% SDS and boiled at 95˚C for 5 minutes to disassemble the Nanodiscs. To remove the large excess of MSP, we then incubated the eluates with Nickel magnetic beads (Cat# B23601; Bimake, Houston, TX) for 1 hour at RT. The MSP remained bound on the Nickel magnetic beads, whereas the proteins contained in the supernatant were precipitated using 100% ice-cold acetone and centrifuged at 15,000g at 4˚C for 10 minutes. The pellets, containing solubilized proteins, were resuspended in Laemmli buffer containing 100 mM DTT, boiled at 95˚C to ensure complete solubilization of the pelleted proteins and then resolved by SDS-PAGE on 4% to 20% Novex gels (Cat# EC60285BOX; Thermo Fisher Scientific, Waltham, MA). Gels were stained with Coomassie brilliant blue (Cat# 1610436; Biorad, Hercules, CA). Gel slices were excised, digested with trypsin, and their protein content analyzed by mass spectrometry at the Arizona Proteomics Consortium.

Samples (DRG and Spinal cord) were harvested two days after intrathecal injections (Fig. 1A). Protein concentrations were determined using the BCA protein assay (Cat# PI23225; Thermo Fisher Scientific). Indicated samples were loaded on 4% to 20% Novex gels (Cat# EC60285BOX; Thermo Fisher Scientific). Proteins were transferred for 1 hour at 120 V using TGS (25 mM Tris pH = 8.5, 192 mM glycine, 0.1% (mass/vol) SDS), 20% (vol/vol) methanol as transfer buffer to polyvinylidene difluoride membranes 0.45 μm (Cat# IPVH00010); Millipore, Billerica, MA), preactivated in pure methanol. After transfer, the membranes were blocked at RT for 1 hour with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% tween 20), 5% (mass/vol) nonfat dry milk, then incubated separately in the primary antibodies CRMP2 (Cat# C2993, Sigma, St. Louis, MO), and βIII-Tubulin (Cat# G712A, Promega) in TBST, 5% (mass/vol) BSA, overnight at 4°C. After incubation in horseradish peroxidase-conjugated secondary antibodies from Jackson ImmunoResearch, blots were revealed by enhanced luminescence (WBKLS0500; Millipore) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 7.1 scanning software by Silk Scientific Inc.

HT-29 and SW620 cells were plated in 10 cm plates at a density of 2 × 10⁶ cells/plate, and HCT-15 cells were plated in 10 cm plates at a density of 10⁶ cells/plate. After attaching overnight, the cells were treated as described for the indicated time. Cells were collected and then lysed with SDS buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA assay kit (Thermo Scientific). Cell lysates were subjected to polyacrylamide gel electrophoresis on Novex gels (Life Technologies), and protein was transferred to PVDF membranes (Life Technologies). Blocking was performed in TBST containing 5% non-fat dry milk or 5% BSA based on the antibody manufacturer’s blocking instructions. The antibody for p62 (#610497) was purchased from BD Biosciences. Antibodies for LC3B (#2775), p-EGFR (#3777), EGFR (#4267), PARP (#9542), ATG7 (#8558), p-mTOR (#5536), and mTOR (#2983) were purchased from Cell Signaling Technology. The antibody for actin (#A5441) was purchased from Millipore-Sigma. The antibody for menin (#A300–105A) was purchased from Bethyl Laboratories. Anti-rabbit and anti-mouse secondary antibodies were purchased from Bio-Rad. The proteins were visualized by detection with Amersham ECL Western blotting detection reagents (GE Healthcare).