Fig. 6
From: Growth inhibition of Saccharomyces cerevisiae by SUMO-specific nanobodies
Smt3p VHHs do not cross react with human SUMO proteins but retrieve Smt3p and Smt3pCTD-tagged GFP from HEK 293T transfectants. (A) Multiple sequence alignment of yeast SUMO (SMT3) and human SUMO variants 1–5 (hSUMO1-5). (B) Native PAGE of VHHs added to human SUMO1 in a threefold molar excess over SUMO1. The migration of hSUMO1 is not affected upon inclusion of the nanobodies. The absence of an additional species that could correspond to a complex indicates the absence of binding. The corresponding SDS PAGE gel is shown below the native PAGE gel. (C) Native PAGE of hSUMO2 showing absence of binding towards Smt3p VHHs similar to (B). (D) VHH1SMT3 fails to recognize human SUMO1 and human SUMO2 in immunoblot. (E) Expression of Smt3p-GFP or GFP tagged with the C terminal region of Smt3p (Smt3pCTD-GFP) in HEK 293T transfectants. SDS PAGE gel of whole cell lysate is shown on the top and the corresponding immunoblots performed with anti-GFP monoclonal antibody and with anti-β actin monoclonal antibody are shown below. (F) Anti-GFP and anti-HA tag immunoblots after retrieval by VHH1SMT3 and VHH2SMT3 of Smt3p tagged GFP from HEK 293T cells. Anti-HA tag indicates the nanobody used for retrieval.
