Ab3742

Human Embryonic Kidney (HEK) 293T cells were cultured in DMEM (Invitrogen). The human monocytic U937 and THP1 cell lines were grown in RPMI (Invitrogen). Media were supplemented with 10% fetal calf serum (Invitrogen) and penicillin/streptomycin (100 U/mL). U937 and THP1 cell lines were differentiated by treatment with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) (300 ng/mL, 24 h). All cell lines were tested mycoplasma-free (Mycoplasmacheck, GATC Biotech). Buffy coats from human healthy donors were obtained from the “Etablissement Français du Sang”. Monocytes were isolated using a CD14+ selection kit (Miltenyi Biotech) and cultured 12 days in DMEM supplemented with 10% Human Serum (inactivated) to generate MDMs. Antibodies used are the following: sheep anti-SUMO1 (Enzo, 1:1000 for WB), rabbit anti-SUMO1 (ab32058, 1:1000 for PLA), rabbit anti-SUMO2/3 (ab3742, 1:1000 for WB, 1:3500 for PLA), mouse anti-SUMO2/3 (ab81371, 1:6000 for PLA), mouse anti-SAMHD1 (ab67820, 1:1000 for WB, 1:5000 for IP, 1:2500 for PLA), rabbit anti-SAMHD1 (ab177462, 1:6000 for PLA), rabbit anti-pT592-SAMHD1 (Cell Signaling #89930, 1:1000 for WB), rabbit anti-actin (Sigma-Aldrich, AA20-33, 1:2500 for WB), anti-HA HRP (Roche Clone 3F10, 1:2500 for WB), Alexa Fluor 488 anti-mouse, Alexa Fluor 594 anti-mouse (INVITROGEN, 1:800 for IF).

Immunohistochemistry was performed on 5 um formalin-fixed and paraffin-embedded brain tissue sections using BOND-MAX Automated Immunohistochemistry Stainer and a BOND Polymer Refine Detection system (Leica Biosystems Melbourne Pty Ltd., Melbourne, Australia). Primary antibodies used were SUMO2/3 1:800 (Abcam, Cambridge, UK, ab3742), anti-Ubiquitin 1:5000 (P4D1; Cell signaling, Danvers, MA, USA) and anti-p62 1:500 (3/P62 LCK LIGAND, BD Bioscience, San Jose, CA, USA). The presence of SUMO2/3 inclusions in both control subjects and subjects with FXTAS was determined blind to the subjects’ genetic information and clinical and pathological diagnosis.