Anti halotag

Immunoblotting was performed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)). Nuclei were lysed in buffer containing 20 mM Tris-HCl pH 7.4, 2.0% NP-40, 500 mM NaCl, 0.25 mM EDTA, 0.1 mM Na₃VO₄, 0.1 mM PMSF, and protease inhibitors. Proteins were resolved using NuPAGE 4–12% Bis-Tris Gel (NPO322BOX; Life Technologies, Carlsbad, CA) and transferred to 0.45 μm Immobilon-FL PVDF membrane (IPFL00010; EMD Millipore Corporation, Massachusetts, MA). Membranes were probed with anti-Cbx7 (ab21873; Abcam, MA) and anti-HaloTag (G9281; Promega, Sunnyvale, CA). After incubating with HRP-conjugated anti-rabbit antibody (NA934V; GE Healthcare, Pittsburgh, PA), proteins were detected using ECL Plus detection reagents (RPN2106; GE Healthcare, Pittsburgh, PA). Membranes were imaged using a ChemiDoc XRS system (BioRad).

Cells were lysed using RIPA buffer (Pierce) supplemented with Complete Mini EDTA-free protease inhibitor (Roche diagnostics GmbH). Cell lysis was further aided by passing cells through 18-gauge needles attached to 2mL syringes. Diluted Laemmli buffer solution (Bio-Rad), mixed with 0.1M DTT (ThermoFisher Scientific), was mixed with equal amounts of supernatant from centrifuged cell solution for western blotting analysis.
Blotting was done using BIORAD mini-protein TGX precast gels and Trans-Blot Turbo 0.2μm (pore size) Mini Nitrocellulose membranes utilising a BIORAD Trans-Blot Turbo System. Blocking was performed in TBST buffer supplemented with 3% BSA (SIGMA life science). Detection was carried out using SuperSignal West Pico PLUS Chemiluminescent Substrate, followed by imaging with a BIORAD ChemiDoc MP imaging system.
The following primary antibodies were used: anti-Pol II, clone F-12 (Santa Cruz, Santa Cruz sc-55492), anti-Dendra2, clone OTI1G6 (ThermoFisher, TA180094), Anti-HaloTag (Promega, G9211) anti-GAPDH, clone 1E6D9 (ThermoFisher, 60004-1-IG). The following secondary antibodies were utilised during western blotting and imaging: m-IgGK BP-HRP (Santa Cruz, sc-516102). Abcam Broad Molecular Weight ladder (ab116028) was used to visualise bands following imaging.

A rabbit polyclonal phospho-serine 316 ATG4B antibody was custom produced by GeneScript by rabbit immunization. pATG4B(Ser316) was diluted 1:1000 in PBST (PBS 0.005% Tween-20) 3% bovine serum albumin (BSA, Sigma-Aldrich, A7906).
Commercially available antibodies and dilutions used are s follows: rabbit ATG4B antibody (Cell Signaling, 5299S, WB dilution 1:1000); mouse monoclonal antibody Anti-HaloTag (Promega, G9211, WB dilution 1:1000); rabbit anti-LC3B antibody (Sigma-Aldrich, L7543, WB dilution 1:1000, immunofluorescence dilution 1:400); rabbit anti-Peroxiredoxin 1 antibody (ABfrontier, LF-MA0031, 1:1000); mouse anti-β-actin (Sigma A1978, WB dilution 1:2000); mouse anti Myc (Millipore, CB430, WB dilution 1:400); rabbit anti-Vinculin (Abcam, AB129002, WB dilution 1:10000); mouse anti-LAMP1 (BD Biosciences, 611042, WB dilution 1:500); mouse anti-GFP (Clontech, 632381, WB dilution 1:1000); mouse biotinylated anti-FLAG (Sigma-Aldrich, F9291, WB dilution 1:1000); and goat anti-GST (GE Healthcare, 27-4577-01, WB dilution 1:1000).

COS-7 cells were harvested 20–24 h after transfection in 300 µL of lysis buffer containing 50 mM HEPES (pH 7.4), 25 mM NaCl, 1 mM EDTA, 1 mM MgCl₂, 0.5% Triton X-100, and protease inhibitors (1 mM PMSF, 0.01 mg/ml Nα-p-tosyl-L-arginine methyl ester, 0.01 mg/ml leupeptin, 0.001 mg/ml pepstatin A, 1 mM DTT). Cell lysates were clarified at 17,000 × g for 10 min before use. Fifty microliters of Protein G Dynabeads (Promega) were resuspended in 200 µL of PBS + 0.02% Tween-20. The beads were incubated with 5 µg of anti-FLAG (Sigma, F4042), anti-GFP (Abcam, ab1218), or anti-HaloTag (Promega, G9281) for 15 min at room temperature. Beads were then washed once with 200 µL PBS + 0.02% Tween-20, once with 200 µL lysis buffer, and incubated with 300 µL lysate for 30 minutes at room temperature. Beads were then washed three times with 300 µL PBS, eluted in 60 µL SDS sample buffer, and analyzed by Western blot.

Recombinant human tPA (Actilyse®) was purchased from Boehringer Ingelheim (Ingelheim am Rhein, Germany). Fetal bovine serum, horse serum, lipofectamine R 2000 reagent, B27 supplement, glutamine, laminin, neurobasal medium, and penicillin/streptomycin were purchased from ThermoFisher (Waltham, Massachusetts, USA). Dulbecco’s modified Eagle’s medium (DMEM), poly-D-lysine, phosphate-buffered saline (PBS), Glycine (Gly), paraformaldehyde, albumin from bovine serum, ammonium chloride (NH4Cl), potassium chloride (KCl), and rabbit polyclonal antibody were purchased from Sigma-Aldrich (St Louis, MO, USA). Tetrodotoxin citrate (TTX), cyanquixaline (CNQX), (2R)-amino-5-phosphonovaleric acid (APV) and bicuculline methiodide (Bic) were purchased from Tocris (Bristol, UK). HaloTag® TMR ligand was purchased from Promega (Madison, Wisconsin, USA). The following primary antibodies were used for immunocytochemistry: mouse monoclonal anti-HaloTag® (dilution 1:1,000; Promega; G9211) rabbit anti-tPA polyclonal antibody (dilution 1:1 500; generous gift from R. Lijnen, Leuven), chicken anti-Microtubule-associated protein 2 (MAP2) polyclonal antibody (dilution 1:8 000; Abcam, Cambridge, UK; ab5392). Secondary fluorescent antibodies (Alexa647; dilution 1:800) were purchased from Jackson Immunoresearch (Bar Harbor, ME, USA).