Figure 2

Association of the various GFP fusion proteins with cells. (a) Jurkat cells were incubated for 1 h with 0.5 μM of FUG (lanes 1 to 3), FP1UG (lanes 4 to 6), FP5UG (lanes 7 to 9) and FP6UG (lanes 10 to 12). After PBS wash, cells were further incubated in RPMI for 4 h. Cells were then washed again with PBS and after collection by centrifugation were lysed in protein loading buffer. Cell extracts (C), together with aliquots of the purified protein (F) and of the first incubation supernatant (S), were loaded onto a SDS protein gel. Immunoblot analysis was carried out using an antibody to GFP. Positions of the signal corresponding to the complete fusion protein (FPG) and of a cleavage product corresponding to GFP (G) are indicated on the left. (b) Proteins FUG, FP1UG, FP2UG, FP3UG and FP4UG were tested as described for panel A, except that only cell extracts were analysed by immunoblot. (c) Activated or non-activated primary lymphocytes were incubated with 0.5 μM of FP1UG, FP1SG, FP5UG or FP5SG for 2 h. Cells were then washed with PBS and lysed in protein loading buffer. Immunoblot analysis was performed as described for panel A and the positions of the signals corresponding to FPSG and FPUG are indicated on the right. (d) and (e) Activated lymphocytes were incubated for 1 h with 0.5 μM of FPG fusion proteins as indicated. This was also done with the FG protein which lacks a PTD motif. After incubation with the proteins, cells were washed in PBS and further incubated in RPMI for 4 h. After a PBS wash, cells were lysed in protein loading buffer. Immunoblot analysis was carried out using a mix of antibody to GFP and to β-actin. Detection of this latter protein was performed to control protein loading in each lane. Revelation was performed with a fluorescent secondary antibody. Positions of signals corresponding to β-actin and FPG are indicated.