Am4300

Cells lysate was prepared by using RIPA buffer followed with SDS-PAGE (Thermo Fisher Scientific) and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at constant 100 V for 1 hr. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 hr at room temperature, the membrane was incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), MYC (Cell Signaling Technology, #9402, 1:1,000), USF2 (Novus, NBP1-92649, 1:2,000), USF1 (Proteintech, 22327–1-AP, 1:2,000), GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), Vinculin (Proteintech, 26520–1-AP, 1:2,000) and CTCF (abcam, ab70303, 1:1,000) at 4°C for 12 hr with gentle shaking. Membranes were washed three times for 30 min and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 hr at room temperature. Blots were washed with TBS-T three times for 30 min and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocol.

Western blotting was performed as described previously [14 (link)]. Briefly, equal amounts of proteins across conditions were separated on NuPAGE Novex Bis-Tris 4–12% Midi gels (Life Technologies) at room temperature and constant voltage. Proteins were transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose) using an iBlot dry blotting system (Life Technologies). After blocking, the membranes were incubated overnight at 4 °C with primary antibodies: mouse monoclonal antibody against glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Thermo Fisher Scientific AM4300, RRID: AB_437392), or rabbit polyclonal antibody against LC3B (1:100; Cell Signaling Technology #2775, RRID: AB_915950, although this antibody was directed against LC3B, cross-reactivity may exist with other LC3 isoforms according to the manufacturer). After extensive washing, the membranes were incubated with peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (1:5000; Jackson ImmunoResearch, Cambridgeshire, United Kingdom) and developed with a chemiluminescent substrate (Western Blotting Luminol Reagent; Santa Cruz Biotechnology, Dallas, TX, USA) in a MicroChemi imaging system (Bioimaging Systems, Upland, CA, USA). Uncropped blots are shown in Figure S1. GAPDH was used as a loading control.

Protein samples were run on 4–20% Tris-Glycine SDS gradient gels and transferred to PVDF membranes using the semi-dry Trans-Blot Turbo system (Bio-Rad). In all, 5% weight/volume milk in PBST was used for blocking and all antibody dilutions. Primary antibodies were incubated on the blots overnight at 4 °C, while horseradish peroxidase-coupled secondary antibodies were on blots for 1 h at room temperature. Clarity Western ECL Substrate (Bio-Rad, 1705060) was used for development, and blots were imaged on a ChemiDoc MP (Bio-Rad). Primary antibodies used: anti-RPL10A (Abcam ab174318, 1:1000 dilution), anti-RPL10A (Santa Cruz Biotechnology sc-100827, 1:1000 dilution), anti-RPL11 (Abcam ab79352, 1:1000 dilution), anti-RPL34 (Abcam ab129394, 1:500 dilution), anti-RPL23A (Bethyl A303-932A-M, 1:2000 dilution), anti-RPL22 (ProteinTech 250021AP), anti-RPS25 (Sigma HPA031801, 1:250 dilution), anti-RPS5 (Abcam ab58345, 1:1000 dilution), anti-GAPDH (Invitrogen AM4300, 1:2000 dilution), anti-β-actin (Cell Signaling 3700 S, 1:2000 dilution). Secondary antibodies used: donkey anti-mouse (GE Healthcare NA931-1ML, 1:5000 dilution) or donkey anti-rabbit (GE Healthcare NA934-1ML, 1:5000 dilution).