Ab70161

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.

Western blot experiment was performed given the previously described methods (Sun et al., 2019 (link)). CD4⁺ T cells, Jurkat T cells, naive CD4⁺ T cells and mouse spleen cells were harvested. The proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) and the protein concentrations were quantified using a bicinchoninic acid (BCA) Protein Assay Kit (TaKaRa). The same amount of protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, Thermo Fisher Scientific) and then transferred into polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked with 5% skimmed milk and then incubated with anti-HUWE1 (ab70161, 1:2,000, Abcam), anti-FoxP3 (ab20034, 1:1,000, Abcam), anti-Ets-1 (ab220361, 1:1,000, Abcam), anti-p-Ets-1 (44-1107G, 1:1,000, ThermoFisher Scientific), anti-MEK1 (ab32576, 1:10,000, Abcam) and anti-GAPDH (ab8245, 1:500, Abcam) overnight at 4°C. The membranes were washed with TBST and incubated with the secondary antibody (ab205718, 1:2,000, Abcam) for 1 h at room temperature. The enhanced chemiluminescence reagents (Thermo Fisher Scientific) and the Versadoc MP 4000 imaging system (Bio-Rad) were performed to visualize protein bands.

Drugs used in this study: Chloroquine (CQ; Sigma-Aldrich, C6628), Arsenic trioxide (ATO; Beijing Chemical, GB673-77) and Parthenolide (BIOMOL, P8522f). Antibodies specific against Caspase3 (9662), cleaved Caspase3 (9661), Pho-p53 (9284), Cdc25 (3652), p21 (2947), CyclinB (12231), Pho-S6K (323), CyclinE1 (20808S), ATG5 (12994), PI3K (4263), Akt (9272), Pho-Akt (13038), Pho-mTOR (5536), Pho-4EBP1 (2855) and BCL-2 (15071) were purchased from Cell Signaling Technologies. Antibodies specific against BAX (sc493), p53 (sc126), catalase (sc50508) and SOD1 (sc11407) were purchased from Santa Cruz. Antibodies specific against HUWE1 (ab70161) and USP7 (ab4080) were purchased from Abcam. Other antibodies were used in this study: CD133 (Immunway, YT5192), GFP (Thermo fisher scientific, MA5-15256), LC3B (Sigma, L7543), mTOR (Genetex, 41731) and GAPDH (Proteintech, 60004-1-Ig). Secondary antibodies used in this study: Peroxidase-conjugated affinity pure goat antimouse IgG, light chain specific (Jackson Immuno Research, 115–035-174), peroxidase-conjugated IgG fraction monoclonal mouse antirabbit, light chain specific (Jackson Immuno Research, 211–032-171).