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. 2013 Feb 11;110(9):3393–3398. doi: 10.1073/pnas.1300328110

Fig. 2.

Fig. 2.

Fluorescence requires deglycosylation and Hrd1. (A and B) HEK293T cells were transfected with the indicated vectors as in Fig. 1, treated with 0.5 µM epoxomicin with or without 20 µM zVAD-fmk for 6 h, and analyzed by FACS. Histograms show Thy1.1+ cells. The geometric mean fluorescent intensity (GMFI) of untreated cells was subtracted from that of treated cells and these values used to determine the percent of epoxomicin-induced fluorescence reported in (B). (C and D) Cells transfected with ZV2 and SS-V1Z or SS-ddV1Z were solubilized in detergent and the lysates immunoprecipitated with the mAb 3E6 and protein A-Sepharose beads. Half of the beads were analyzed for fluorescence using a plate reader (C), and the other half were separated by SDS/PAGE, transferred to Immobolin-P, and blotted with anti-GFP-biotin (D). (E and F) Cells were transfected with the indicated vectors and DN Hrd1 or vector control. Thy1.1+ cells were analyzed by FACS after treatment for 6 h with 0.5 µM epoxomicin. Results in B and F are the mean+/−SEM of at least three independent experiments (***P < 0.001). Results in C and D are representative of at least three independent experiments. ND, not determined.