Figure 6. Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.

(A) LC-MS/MS quantitation of UDP-GlcNAc in post-nuclear supernatants (PNS), vesicular, and cytosolic fractions of kifunensine treated and untreated Jurkat T cells. (B) PNS (input) from kifunensine treated and untreated Jurkat T cells was used for Golgi enrichment via anti-TGN46 immuno-isolation followed by blotting for the Golgi compartment markers GM130 (cis), GS27 (medial/trans), and Syntaxin6 (trans). (C) LC-MS/MS quantitation of UDP-GlcNAc in anti-TGN46 bound and unbound fractions from kifunensine treated and untreated Jurkat T cell PNS. (D) PNS of Jurkat T cells treated for 0, 15, and 30 min with the Golgi disruptor Brefeldin A were used for anti-TGN46 immuno-isolation followed by blotting for B3GNT2 and the GM130 (cis), GS28 (cis/medial), and TGN46 (trans) Golgi markers. (E) Quantitation of D, (F) LC-MS/MS quantitation of UDP-GlcNAc in anti-TGN46 immuno-isolates from Jurkat cells treated with Brefeldin A for 15 min. (G) LEA flow cytometric analysis of Jurkat T cells pre-treated with nocodazole where indicated for 45 min, followed by swainsonine where indicated for 5 hr. (H) Jurkat T cells that were treated with nocodazole +/- pyrrophenone as indicated for 45 min were treated with or without swainsonine for 5 hr and then analyzed for LEA and ConA binding by flow cytometry. Shown is the ratio of LEA MFI to ConA MFI for each condition. NS, not significant; *p<0.05; **p<0.01; ***p<0.001; (unpaired one-tailed (A, C, F and G) or two-tailed (H) t-test with Welch’s correction and Bonferroni correction (H)). Data show one experiment representative of at least three independent experiments except F which shows combined data from two independent experiments. Error bars indicate mean ± s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.14814.017

Figure 6—figure supplement 1. Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.

(A–C) LC-MS/MS intensity plots of 1.11 ng/ml UDP-GlcNAc standard (A), and untreated (B) and 0.1% triton-X treated (C) Jurkat T cell vesicular fraction. (D) LC-MS/MS quantitation of UDP-GlcNAc levels in untreated versus 0.1% triton-X or 50 mM UMP treated Jurkat T cell vesicular fractions. (E) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine for an additional 5 hr, at which time cells were analyzed for LEA and ConA binding by flow cytometry. The y-axis indicates percent reversal of the swainsonine-induced change, where 0 corresponds to lectin binding in untreated cells and 100 to swainsonine treatment alone. (F–H) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine where indicated for an additional 5 hr, after which cells were analyzed for LEA, ConA, and L-PHA binding by flow cytometry. NS, not significant; **p<0.01; ***p<0.001; (unpaired two-tailed t-test with Welch’s (D and F–H) and Bonferroni correction (F–H). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.14814.018