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Figure 4 | BMC Biotechnology

Figure 4

From: Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains

Figure 4

Negative effect of the SUMO-1 diglycine motif on detection of intracellular fusion proteins. (a) Schematic representation of fusion protein associating the FLAG-PTD-SUMO-1 module with the HA epitope. Using FPSG vectors, the GFP moiety was deleted and replaced with the HA epitope coding sequence, giving vectors expressing FPSH proteins. (b) Activated lymphocytes were incubated for 2 h with 2 μM of the different FPSH proteins, then washed with PBS and lysed in RIPA buffer. Supernatant (S) or RIPA extracts (RE) were analysed by immunoblot using a monoclonal antibody to HA (c) Activated lymphocytes were incubated for 2 h without (lane 3) or with 1 μM of either FP1SH (lane 1), FP5SH (lane 2), SHPR142 (lane 4) or SHPR190 (lane 5). Cells were then washed with PBS and lysed with RIPA buffer. Cell extracts were analysed by immunoblot with monoclonal antibodies to SUMO-1 (top panel) and to β-actin (lower panel). Signals corresponding to SUMO-1 fusion proteins and to β-actin are indicated on the right. (d) Schematic representation of the SHP and SGHP proteins. Using the vector expressing SHPR-190, the AR motif was restored to the wild type GG sequence occurring at the end of SUMO-1, giving SGHP. (e) Activated lymphocytes were incubated with SGHP, SHPT8 or SHPR190 for 2 h and washed in PBS. RIPA extracts were then made and analysed by immunoblot using a monoclonal antibody to SUMO-1.

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