Anti sumo2 3

Cells were lysed in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.1% Nonidet P-40) supplemented with protease inhibitors (Roche, Boulogne-Billancourt, France) and 5 mM N-ethylmaleimide (Sigma). Proteins were separated on SDS/PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Velizy-Villacoublay, France), blocked with 5% non-fat milk in PBS containing 0.1% Tween-20. Membranes were then probed overnight at 4°C with the relevant primary antibodies: anti-SUMO1 (Sigma), anti-SUMO2/3 (Sigma), anti-LC3 (Sigma), anti-SAE1 (Abcam, Paris, France), anti-SAE2 (Abcam), anti-UBC9 (Abcam), anti-PIAS3 (Cell Signaling Technology, Saint Quentin Yvelines, France), anti-p62 (Cell Signaling Technology), anti-β-actin (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology). After washes, membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology), and blots were detected using the Enhanced Chemiluminescence Detection kit (Amersham Biosciences, Charfent, UK).

The pCDH-Strep-ALKBH5 expression plasmid was generated by cloning the corresponding coding sequence into pCDH-Strep vector. All the pCDH-Strep-ALKBH5 K/R (lysine to arginine) or S/A (serine to alanine) mutants were derived from pCDH-Strep-ALKBH5 by site-directed mutagenesis. All expression plasmids for the SUMO systems were kindly provided by Dr. Jiemin Wong's lab. Antibodies used in this study were listed as follows: anti-m⁶A (Synaptic Systems# 202003), anti-γ H2A.X (CST#9719S), anti-γ H2A.X (Thermo Fisher#MA1-2022), anti-ALKBH5 (Sigma#HPA007196), anti-ALKBH5 (Thermo Fisher #703570), anti-FTO (Sigma#SAB2106776), anti-METTL3 (Sigma#SAB2104747), anti-METTL14 (Sigma#HPA038002), anti-SUMO-1 (Thermo Fisher #33–2400), anti-SUMO-2/3 (CST#4971P), anti-ERK (CST#9102S), anti-p-ERK (CST#9106S), anti-JNK (CST#9252S), anti-p-JNK (CST#4671S), anti-phophoserine (Sigma#P5747), anti-phophotyrosine (Sigma#SAB5200015), anti-Strep (Sigma#SAB2702216), anti-Annexin V (Thermo Fisher #17800774), anti-Actin (CST#8457S), anti-Tubulin (CST#2146S), anti-Lamin A/C (CST#4777S), anti-HA (CST#2362), anti-Flag (Sigma#F1804), anti-IGF2BP2 (CST#14672S), anti-eIF3A (CST#3411S).