(A) O-GlcNAc transferase (OGT) uses the nucleotide-sugar donor UDP-GlcNAc to add O-GlcNAc to protein substrates. O-GlcNAcase (OGA) removes O-GlcNAc moieties. 5SGlcNAc and Thiamet-G are specific small molecule inhibitors of OGT and OGA, respectively. (B) 293T cells were treated with DMSO vehicle or GlcNDAz (48 hr) and UV light (or not) as indicated, and lysates were prepared in denaturing buffer and analyzed by IB. O-GlcNAc-mediated protein-protein interactions manifest as high molecular weight GlcNDAz-crosslinked complexes (labeled). Heavily glycosylated nucleoporin-62 is a positive control, whereas unglycosylated tubulin is a negative control. Vimentin IB was performed with the D21H3 antibody. (C) 293T cells were transfected with OGT-myc or OGA-myc constructs, as indicated, and subjected to GlcNDAz crosslinking as above. Crosslinked and uncrosslinked endogenous vimentin species were detected in lysates made in denaturing buffer by IB (D21H3 antibody). (D) 293T cells were subjected to GlcNDAz crosslinking as above. Then, soluble (disassembled) and insoluble (assembled) vimentin populations were separated by differential extraction, as described (Herrmann et al., 2004). Low, low ionic strength buffer. High, high dil., high ionic strength buffer (loaded both as-is and diluted, as recommended (Herrmann et al., 2004)). 8M urea extracts fully assembled IFs. Crosslinked and uncrosslinked vimentin species were detected by IB (D21H3 antibody). (E) 293T cells were treated with GlcNDAz for 48 hr, treated with 1% IDPN or DMSO vehicle for 30 min, and exposed to UV. Then, cells were subjected to differential extraction, as above, and analyzed by IB (D21H3 antibody). Note that the uncrosslinked vimentin band appears in the 8M urea fraction of IDPN-treated cells because IDPN treatment collapses vimentin IFs into insoluble aggregates (see Figure 1—figure supplement 2G) (Durham, 1986).