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. 2019 Feb 11;77(1):ftz007. doi: 10.1093/femspd/ftz007

Figure 5.

Figure 5.

eEF1A1 reduces K48-linked EBI3 ubiquitination in iH37Rv-treated macrophages. (A) EBI3 expression was reduced in eEF1A1-silenced macrophages upon iH37Rv stimulation. RAW 264.7 cells were transfected with eEF1A1 shRNA. After 24 h of transfection, cells were stimulated with iH37Rv for 6 h. Upper panel, after 24 h of transfection, eEF1A1 expression was determined by immunoblot analysis. Lower panel, after 6 h of iH37Rv stimulation, EBI3 expression was determined in eEF1A1-slienced cells. (B) eEF1A1 inhibited K48-linked ubiquitination of EBI3 in iH37Rv-treated cells. RAW 264.7 cells were transfected with eEF1A1 shRNA and stimulated with iH37Rv. Supernatant from the cell lysate was incubated with anti-EBI3 antibody followed by incubation with protein A/G magnetic beads. The precipitated samples were subjected to immunoblot analysis with anti-EBI3 antibody and K48-linkage specific polyubiquitin antibody. (C) eEF1A1-silienced RAW 264.7 cells were treated with proteasome inhibitor MG132 (2 nM) prior to iH37Rv treatment. The EBI3 production were measured by immunoblot analysis with anti-EBI3 antibody.