Ab179466

Cells were seeded directly in presence or absence of IFN-γ/TNF-α on glass slices in 6-well plates to a confluency of approximately 50%. Twenty-four hour later, cells were washed in PBS and fixed for 10 min in 4% formaldehyde /PBS. After two washing steps in PB buffer (PBS, 3% BSA), cells were permeabilized for five minutes in PBGT buffer (PBS, 0.2% Triton X-100, 20 mM Glycine and 3% BSA). After two subsequent washing steps with PBG buffer (PBS, 20 mM Glycine, 3% BSA), cells were incubated for 2 hours in the dark with the following primary antibodies, diluted in PBG: anti-SUMO-1-21C7 mouse monoclonal antibody^{55 (link)} (1:50), anti-PML (Abcam, ab179466, rabbit mAb, 1:500) and Phalloidin Alexa Fluor® 647 (Invitrogen, A22287, 1:250). Nuclear staining was performed with Nuclear Green DCS1 (Abcam, ab138905, 1:2000). Slices were carefully washed four times with PB buffer and then incubated for one hour in the dark with the secondary antibodies anti-mouse-Alexa 488 (Life Technologies, 1:250) or anti-rabbit-Alexa 568 (Life Technologies, 1:250). After four wash steps with PBS, slices were imbedded with fluorescent mounting medium (Dako, E3023) and imaged on a Leica TCS SP5 II laser scanning microscope using a ×63/1.4 NA oil-immersion objective (Leica).

Cell and tissue were lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails (NCM Biotech, China) for 30 min at 4°C and then centrifuged at 13000 × g for 30 min. Protein supernatants were separated by 7.5% (wt/vol) SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, USA). After blocking with 5% milk for 1 h at RT, the membranes were incubated with the primary antibodies at 4°C overnight and then with secondary antibodies conjugated to a fluorescent tag (Invitrogen, USA). The band signals were visualized by an Odyssey Infrared Imaging System (LI-COR, USA). The following primary antibodies were used in the study: anti-ALKBH3 (ab251697, Abcam, USA), anti-LDHA (19987–1-AP, Proteintech, China), anti-LDHB (14824–1-AP, Proteintech, China), anti-SP100 (PA5-53476, ThermoFisher, USA), anti-PML (ab179466, Abcam, USA), anti-YTHDF1 (57530, CST, USA), anti-Pan Kla (1401RM, PTM, China), anti-H3K18la (1406RM, PTM, China), anti-Flag (2368S, CST, USA), anti-ACTB (3700, CST, USA) and anti-Histone H3 (4499, CST, USA). Unprocessed western blot figures were performed in Supplementary Figures S14 and S15.

Cells were lysed directly in the cell culture plates with 3× SDS loading buffer (B7703S; New England BioLabs, Ipswich, MA, USA). After boiling at 95 to 100°C for 10 min, the samples were removed from the BSL3 facility for further processing. Cell lysates were briefly sonicated (10 to 12 s) to reduce sample viscosity. After centrifugation, supernatants were collected and boiled at 95 to 100°C for 5 min. An equal volume of samples (25 to 30 μL) was separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Nonspecific binding was blocked by immersing the membrane in Tris-buffered saline-Tween (TBST) blocking solution containing 5% skim milk powder. After blocking, the membranes were incubated with the primary antibody overnight at 4°C followed by the appropriate secondary antibody for 1 h at room temperature. Proteins were detected using enhanced chemiluminescence (ECL). The primary antibodies used were anti-NRF2 (ab62352; Abcam, Cambridge, United Kingdom), anti-PML protein (ab179466; Abcam, Cambridge, United Kingdom), and anti-β-actin as the loading control (A1978 [Sigma-Aldrich, St. Louis, MO, USA] and sc-47778 HRP [Santa Cruz Biotechnology, Dallas, TX, USA]).

Cells were lysed directly in the cell culture plates with 3× SDS loading buffer (B7703S; New England BioLabs, Ipswich, MA, USA). After boiling at 95 to 100°C for 10 min, the samples were removed from the BSL3 facility for further processing. Cell lysates were briefly sonicated (10 to 12 s) to reduce sample viscosity. After centrifugation, supernatants were collected and boiled at 95 to 100°C for 5 min. An equal volume of samples (25 to 30 μL) was separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Nonspecific binding was blocked by immersing the membrane in Tris-buffered saline-Tween (TBST) blocking solution containing 5% skim milk powder. After blocking, the membranes were incubated with the primary antibody overnight at 4°C followed by the appropriate secondary antibody for 1 h at room temperature. Proteins were detected using enhanced chemiluminescence (ECL). The primary antibodies used were anti-NRF2 (ab62352; Abcam, Cambridge, United Kingdom), anti-PML protein (ab179466; Abcam, Cambridge, United Kingdom), and anti-β-actin as the loading control (A1978 [Sigma-Aldrich, St. Louis, MO, USA] and sc-47778 HRP [Santa Cruz Biotechnology, Dallas, TX, USA]).

Chemicals: sodium arsenite (Carlo Erba); Z‐Leu‐Leu‐Leu‐al (MG132; C2211, Sigma‐Aldrich); Cycloheximide (C7698, Sigma‐Aldrich); ML-792 (HY-108702, Medchemexpress); DAPI (SC3598, Santa Cruz Biotechnology).
Primary antibodies: PML (ab179466 and ab53773, abcam 1:100); TIAR (610352, BD Biosciences, 1:100); G3BP1 (611126, BD Biosciences, 1:100); neuron-specific class III beta-tubulin/TuJ1 (60052, Stemcell Technologies, 1:500); Choline Acetyltransferase/ChAT (SAB2500236, Merck, 1:75); polyUb proteins (FK1; BML‐PW8805‐0500, Enzo, 1:100); TUBA4A (T6074, Merck, 1:1000); SUMO1 (ab49767 and ab219724, abcam, 1:100); SUMO2/3 (ab3742, abcam, 1:100 and 10947-1-AP, proteintech 1:100).
Secondary antibodies: mouse IgG HRP linked whole ab (NXA931, GE Healthcare, 1:5000); rabbit IgG HRP linked whole ab (NA934, GE Healthcare, 1:5000); Alexa Fluor™ 594 Azide (A‐10270, Thermo Scientific, 10 μM); Donkey anti‐Mouse IgG (H + L), Alexa Fluor® 594 (A‐21203, Thermo Scientific, 1:1000); Donkey anti‐Mouse IgG (H + L), Alexa Fluor® 488 (A‐21202, Thermo Scientific, 1:1000); Donkey anti‐Rabbit IgG (H + L), Alexa Fluor® 594 (A‐21207, Thermo Scientific, 1:1000); Donkey anti‐Rabbit IgG (H + L), Alexa Fluor® 488 (A‐21206, Thermo Scientific, 1:1000).