Sc 8017

Western blotting was carried out as described in Text S1. Ubiquitin epitopes were detected using an anti-ubiquitin antibody (P4D1, RRID:AB_628423; diluted 1:2,000 [sc-8017; Santa Cruz Biotechnology]). Membranes were stained with 0.15% (wt/vol) Ponceau S (40% methanol, 15% acetic acid [P3504; Sigma]) as a loading control.

Immunoblotting was carried out as previously described using antibodies against HUWE1, (ab70161, Abcam), c-MYC (ab32072, Abcam), GAPDH (ab181602, Abcam) and ubiquitin (sc-8017, Santa Cruz, Heidelberg, Germany) and secondary antibodies anti-mouse and anti-rabbit (DAKO, Cambridgeshire, UK). Ubiquitinated proteins were isolated using UbiQapture-Q (Enzo Life Sciences, Exeter, UK) to bind both mono- and poly-ubiquitinated proteins independent of lysine chain linkage, or tandem ubiquitin binding entity (TUBE) antibodies specific for either K48 or K63 linkages (UM607 and UM604, Life Sensors, Malvern, USA), according to the manufacturer’s instructions. A proportion (25 μg) of total cell lysate was retained for use as an input control and 250 μg of remaining lysate was bound to the appropriate affinity matrix to capture ubiquitinated proteins. Input controls and protein eluted from the matrix were subsequently analyzed by immunoblotting. Blots were visualized using WesternBright^™ ECL horseradish peroxidase substrate (Advansta, UK), scanned into the G:BOX gel doc system (Syngene, Cambridge, UK) and analyzed using GeneTools.

Cells were seeded in 96-well black plates. Treatments were performed as described previously. The fixation step protocol was followed [25 (link)]. After permeabilization, the cells were incubated with primary antibodies against CSTD (D-7) (sc-377299, Santa-Cruz Biotechnology, Paso Robles, CA, USA, 1:200), LAMP1 (H4A3) (sc-20011, Santa-Cruz Biotechnology, Paso Robles, CA, USA, 1:200) and ubiquitin (P4D1) (sc-8017, Santa-Cruz Biotechnology, Paso Robles, CA, USA, 1:200) for 1–2 h at room temperature and then incubated with Alexa Fluor^® 568 (A11004)- or 488 (A11008)-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Image quantification was conducted with Image J software (National Institute of Health, Bethesda, MD, USA).

Western blot was conducted according to previously described.¹⁷ RIPA buffer (Beyotime, China) containing protease inhibitor (Thermo Fisher Scientific, USA) was used to isolate total proteins. Then, the concentration of proteins was evaluated by BCA Protein Assay kit (Thermo Fisher Scientific). Sodium dodecylsulphonate (SDS) polyacrylamide gel electrophoresis was used to separate proteins, followed by electroblotting (Millipore, USA) which can transfer proteins onto a polyvinylidene fluoride (PVDF) membrane. Next, PVDF membranes were incubated with primary antibodies for a night at 4°C. The following antibodies were employed to examine target proteins: anti‐OTUB1 monoclonal antibody (1:500; ab175200, Abcam), anti‐EYA1 polyclonal antibody (1:500, ab194448, Abcam) and anti‐ubiquitin monoclonal antibody (1:500, sc8017, Santa Cruz). After incubation with secondary antibody (Proteintech, China) for 1 h at 37°C, protein band intensity was evaluated through Quantity One software (Bio‐Rad, USA).

Cell pellets were resuspended in Laemmli sample buffer (BioRad), and Western blots were performed as described previously (McClintock et al., 2006 (link)). The membranes were incubated with primary antibodies: anti-lamin A/C [kindly provided by Dr. N. Chaudhary (1/5000) (Chaudhary & Courvalin, 1993 (link)), anti-progerin antibody (clone S9, 0.1 μg mL⁻¹) (McClintock et al., 2007 (link)), anti-prelamin A antibodies (sc-6214, Santa Cruz Biotechnology, 1/1000), anti-proteasome S20 subunit C2 (ab22665, Abcam, 1/000), anti-Hsp27 (ab2790, Abcam, 1/2000), anti-ubiquitin (sc-8017, Santa Cruz Biotechnology, 1/3000), anti-LC3B (Sigma-Aldrich, 1/4000), anti-53BP1 (A300-272A, Bethyl, 1/1000)], anti-FHL-1 (sc-133580, Santa Cruz Biotechnology, 1/1000) anti-Rad51 (NBP2-32622, Novus Biological, 1/1000) anti-β-actin (Sigma-Aldrich, 1/5000) and anti-β-tubulin (Thermo Fisher, 1/2000). Then washed and incubated with a corresponding secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). Proteins were visualized using a chemiluminescence detection system (ECL substrate; BioRad). Signals were analyzed with image lab software (BioRad). Protein signals were quantified by normalizing to β-actin as indicated.

WB was performed according to the previously reported methods, with some modifications [21 (link)]. Cells were harvested in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) that was supplemented with complete protease inhibitors (Sigma, Darmstadt, Germany). Cell lysates were separated on 10% SDS-PAGE and blotted onto the NC membrane. The membrane was then blocked in 5% nonfat milk and incubated with the primary antibodies overnight at 4 °C in the following dilutions: anti-G3bp1 (1:3000, ab181150, Abcam, Cambridge, UK), anti-G3bp1 (1:3000, ab56574, Abcam, Cambridge, UK), anti-G3bp2 (1:3000, NBP1-82977, Novus biologicals, Abingdon, UK), anti-Ubiquitin (1:1000, sc-8017, Santa Cruz, Dallas, TX, USA), anti-HSP70 (1:10000, ab45133, Abcam, Cambridge, UK), anti-VCP (1:3000, ab109240, Abcam, Cambridge, UK), anti-LC3 (1:1000, A19665, ABClonal, Wuhan, China), and anti-GAPDH (1:10000, KC-5G5, Kangchen Biotech, Shanghai, China). After the membranes were washed with 0.1% PBST, HRP-conjugated anti-mouse (7076S, CST, Danvers, MA, USA) or anti-rabbit (7074S, CST, Danvers, MA, USA) antibody was used as the secondary antibody for 1 h at room temperature. The signals were detected by using the Western ECL Substrate (170-5060, Bio-Rad, Irvine, CA, USA). The images were visualized by using the Bio-Rad ChemiDoc XRS+ system.

293T cells were seeded at a density of 40% on a 100-mm dish. After cell attachment, the cells were transfected with expression vectors using FuGENE6, and after 48 h, transfected 293T cells were washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science). Cell extracts were incubated with anti-FLAG M2 agarose (Sigma-Aldrich) for 2 h at 4°C. After the beads were washed 3 times with 1 ml of TBS buffer (pH 7.6), the FLAG-tagged proteins bound to the beads were eluted by boiling in Lane Marker Sample Buffer (Thermo Fisher Scientific). Samples were then subjected to SDS-PAGE, and detected by western blot. The following antibodies were used: anti-LSD1 (ab17721, Abcam, Cambridge, UK), anti-CoREST (ab32631, Abcam), anti-HDAC1 (sc7872, Santa Cruz Biotechnology, Dallas, TX), anti-HDAC2 (sc7899, Santa Cruz Biotechnology), anti-Ubiquitin (sc8017, Santa Cruz Biotechnology), anti-H3K4me1 (ab8895, Abcam), anti-H3K4me2 (ab32356, Abcam) and anti-histone H3 (ab1791, Abcam).