FIG 4 .
LTIIa delivers cargo (βlac) more efficiently into GD1b-enriched Neuro-2a cells than GM1a-enriched Neuro-2a cells. (A) Neuro-2a cells were loaded with 10 µg/ml of ganglioside GD1b or GM1a in DMEM with 0.5% FBS at 37°C for 3 h. Cells were washed and incubated with 40 nM βlac-LTIIa or LTIIa at 37°C for 60 min. Cells were loaded with CCF2-AM at RT for 30 min, followed by IF staining using anti-HA antibody (red). Uncleaved CCF2 is shown in green, and cleaved CCF2 (CCF2C) is shown in cyan. (B) Cleavage of substrate CCF2 was quantified using the CCF2C/CCF2/HA ratio of fluorescent intensities. (C) Neuro-2a cells were loaded with GD1b and then incubated with 40 nM βlac-LTIIa, βlacnull-LTIIa, or LTIIa at 37°C for 60 min alone or with 0.1 µg/ml of brefeldin A (BFA). Cells were washed and were loaded with CCF2-AM at RT for 30 min. Cleavage of CCF2 was quantified using the CCF2C/CCF2/HA ratio of fluorescent intensities. Data were analyzed by two-tailed Student’s t test. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Bar, 20 µm.