Skip to main content
. Author manuscript; available in PMC: 2018 Oct 12.
Published in final edited form as: Cell Rep. 2018 Sep 18;24(12):3296–3311.e6. doi: 10.1016/j.celrep.2018.08.057

Figure 3. S100A8 Is a Substrate of RNF5.

Figure 3.

(A) Immunoprecipitation and immunoblot analysis of the RNF5-S100A8 interaction in HEK293T cells ectopically expressing Flag-tagged RNF5 or empty vector (EV) and V5-tagged S100A8.

(B) Immunoprecipitation and immunoblot analysis of HEK293T cells co-expressing V5-S100A8, HA-ubiquitin (HA-Ub), plus Flag-RNF5. Cells were treated withMG132 (10 μM) for 4 hr prior to lysis.

(C) Anti-S100A8 immunoprecipitation and anti-Ub immunoblot of MODE-K cells transfected with EV or RNF5-targeting shRNA and treated with MG132 (10 μM) for 4 hr before lysis.

(D) Immunoblot analysis of HEK293T cells expressing Flag-EV or Flag-RNF5 and treated with medium or 10 μM MG132 for 4 hr before lysis.

(E) Immunoblot analysis of MODE-K cells expressing EV or shRNF5. Cells were treated with cycloheximide (CHX) as indicated (n = 3).

(F) Immunoprecipitation and immunoblot analysis of the interaction between endogenous RNF5 with S100A8 analyzed in MODE-K cells treated with MG132 (10 μM) for 4 hr.

(G) Immunoprecipitation and immunoblot analysis of cell lysates prepared from MODE-K cells treated with 10 ng/mL TNF-α for the indicated times followed by MG132 (10 μM) for 4 hr before lysis.

Data are representative of three independent experiments. Graphs show mean ± SEM.