Ab83497

The western blot analysis for the HTT protein (17/68Q tract) was performed as previously described (Fiszer et al., 2011 (link)). Briefly, 30 μg of total protein was run on a Tris-acetate sodium dodecyl sulfate (SDS)-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad, Hercules, CA, USA) at 130 V in an ice-water bath. Subsequently, the proteins were wet-transferred to a nitrocellulose membrane (Sigma–Aldrich). All of the immunodetection steps were performed using the SNAPid system (Millipore). The primary antibodies anti-huntingtin (1:1000, MAB2166, Millipore) and anti-plectin (1:1000, ab83497, Abcam, Cambridge, UK) and secondary antibodies anti-mouse HRP-conjugate (1:2000, A9917, Sigma–Aldrich) and anti-rabbit HRP-conjugate (1:2000, 711-035-152, Jackson ImmunoResearch) were used in a PBS/0.1% Tween-20 buffer containing 0.25% non-fat milk. The immunoreaction was detected using WesternBright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and were quantified using Gel-Pro Analyzer.

Dynabeads™ MyOne™ Streptavidin T1 (Thermo Fisher) were coated with biotinylated PCS1, 2 and control peptoid PC462^{6 (link)}. Cells were dissociated using Dissociation Buffer or 1x accutase (Sigma). The beads were equilibrated with 2 million cells and incubated for 10 min at RT with gentle shaking. Bead-bound cells and non-bound cells were separated by magnet and counted by Cellometer Mini (Nexcelom). A fraction of each bead-bound sample was then plated for visualization by microscope and then the remaining cells were harvested for RT-qPCR or used in Clonogenicity assay. For the antibody-based magnetic bead binding assay, biotinylated plectin antibody (Bioss Antibodies, Woburn, MA), was used in place of the biotinylated compounds. For the competitive binding assay (Fig. 3E), 1 µg of c-terminal-targeting plectin antibody (ab83497 or ab32528, Abcam, Cambridge, MA) was added to the incubation between beads and cells.

A total of 30 μg of protein was resolved on a Tris-acetate SDS-polyacrylamide gel (3–8%, NuPAGE^TM, Invitrogen, Carlsbad, CA, USA) in Tris-Acetate SDS Running buffer (Novex, Carlsbad, CA, USA) at 170 V at 4°C. After electrophoresis, the proteins were wet-transferred overnight to a nitrocellulose membrane (Sigma–Aldrich). The primary antibodies, namely, anti-huntingtin (1:1000, MAB2166, Millipore, Burlington, MA, USA) and anti-plectin (1:1000, ab83497, Abcam, Cambridge, UK), and the secondary antibodies, namely, the anti-mouse HRP conjugate (1:2000, A9917, Sigma–Aldrich) and anti-rabbit HRP conjugate (1:2000, 711-035-152, Jackson ImmunoResearch, West Grove, PA, USA) were used in a TBS/0.1% Tween-20 buffer containing 2.5% non-fat milk. The immunoreaction was detected using Western Bright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and quantified using Gel-Pro Analyzer (Media Cybernetics).