Anti strep

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).

After SDS-PAGE, gels were transferred onto polyvinylidene difluoride (PVDF) membranes using an iBlot 2 Dry Blotting System (Invitrogen). The membranes were blocked for one hour in TBS buffer (200 mM NaCl, 20 mM Tris pH 7.5) supplemented with 0.1% Tween-20 and 4% milk powder (anti-mScarlet and anti-FLAG) or 4% bovine serum albumin (anti-mNEON and anti-Strep). The primary antibodies (anti-mNEON from Chromotek diluted 1:500, anti-mScarlet from Chromotek diluted 1:2000, anti-Strep from IBA diluted 1:1000, and anti-FLAG from Sigma-Aldrich diluted 1:1000) were incubated for two hours at room temperature under gentle agitation. The PVDF membranes were washed three times in TBS-0.01% Tween-20 for 10 minutes. The secondary antibody, IgG conjugated with Alkaline Phosphatase (Sigma) was diluted 1:5000 and incubated for 1 hour at room temperature under gentle agitation. The PVDF membranes were washed again twice in TBS-0.01% Tween-20, and once in TBS buffer. The membranes were developed using BCIP/NBT alkaline phosphatase substrate (Sigma).