Mini gel tank system

Protein samples were loaded into a Bolt™ 4–12% Bis-Tris Plus Gel (Invitrogen™) with MOPS 1X buffer supplemented with Bolt™ Antioxidant (0.25%), and a Mini Gel Tank system (Invitrogen™) was used. Transference to nitrocellulose membranes was performed using the Mini Blot Module (Invitrogen™). For protein detection, membranes were blocked for 1 h with blocking solution (TBS + 0.1% Tween-20 (TBS-T) + 5% of non-fat powdered milk) and incubated overnight at 4 °C with the following primary antibodies: rabbit α-InlA 1:5000 (Cusabio) or rabbit α-EF-Tu 1:40000 (Abcam) in TBS-T. The secondary antibody goat α-rabbit IgG-HRP 1:20,000 (Sigma) was incubated for 1 h at 4 °C. Detection was performed by chemiluminescence using Western Lightning® Plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer) in iBright™ CL1500 Imaging System (Supplementary Fig. 5). Images were quantified using Fiji software.

OGT: H460 cells were lysed in Pro-Prep protein extraction solution (INtRON Biotechnology, Gyeonnggi-do, Korea), miR-7-5p transfection after 72 h. An equal amount of protein was dissolved in 8% SDS-PAGE gels (Laemmli, 1970). The primary antibodies used for the analysis were mouse anti-OGT (1:1000 cell signaling) and b-actin antibodies (1:2000; Santa Cruz Biotechnology)
O-GlcNAc: HeLa cells were lysed in Pro-Prep protein extraction solution (INtRON Biotechnology, Gyeonnggi-do, Korea). The miR-7-5p was transfected after 72 h. The Mini Gel Tank System (Invitrogen) was used for protein sample gel loading, and an iBlot2 (Invitrogen) membrane transfer was done. The primary antibodies used for the analysis were mouse anti- O-GlcNAc (1:1000 cell signaling) and b-actin antibodies (1:2000; Santa Cruz Biotechnology)

Full length antibody, purified F(ab)'2 and purified Fc fragments were diluted in NuPAGE LDS sample buffer (#NP0007, Invitrogen), heated at 70 °C for 10 min and loaded onto Bolt^TM 4-12% Bis-Tris gels (#NW04120BOX, Invitrogen). Electrophoresis was run on the Mini Gel Tank system (Life Technologies) at 200V constant voltage in NuPAGE MES SDS running buffer (#NP0002, Invitrogen). Gels were fixed and stained with Coomassie brilliant blue R-250 (#1610436, Bio-Rad).