American Society of Hematology

Figure 2

$Figure 2. Binding and association of CD300a-Ig to PE and PS. (A) Starved PBMCs from healthy donors were left untreated (alone) or preincubated with 2 different divalent cation chelators (EDTA and EGTA). The binding of LAIR-1-Ig or CD300a-Ig to the 7-AAD positive cells was assessed and the mean fluorescence intensity (MFI) values are plotted. Data shown is representative of 3 independent experiments. (B) Liposomes of specified compositions (x-axis) were prepared and coupled to a L1 biosensor. See the supplemental Methods for specifics of liposome composition. The binding of LAIR-1-Ig (white bars) and CD300a-Ig (black bars) was analyzed by allowing the proteins to pass through the L1 sensor. The binding (resonance units or RU) for plateau values is shown. The RU are plotted after subtracting from the buffer control. Bar graph represents the average ± SEM. Results shown are from 8 independent experiments. (C) LAIR-1-Ig and CD300a-Ig were immobilized onto a CM5 sensor and a series of liposomes with different compositions (x-axis) were allowed to pass through the sensor and the binding analysis of liposomes to proteins was analyzed. See supplemental Methods for specifics of liposome composition. The binding (RU) for plateau values is shown. The RU are plotted after subtracting from the buffer control. Bar graph represents the average ± SEM. Results shown are from 2 independent experiments. (D) Liposomes of different compositions were incubated with CD300a-Ig fusion protein and allowed to associate. After 40 minutes of incubation the supernatants (S) and pellet (P) fractions were separated by ultracentrifugation and analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) analysis. The prestained protein markers (M) are also represented on the protein gel. Data shown are representative of 3 independent experiments.$

Binding and association of CD300a-Ig to PE and PS. (A) Starved PBMCs from healthy donors were left untreated (alone) or preincubated with 2 different divalent cation chelators (EDTA and EGTA). The binding of LAIR-1-Ig or CD300a-Ig to the 7-AAD positive cells was assessed and the mean fluorescence intensity (MFI) values are plotted. Data shown is representative of 3 independent experiments. (B) Liposomes of specified compositions (x-axis) were prepared and coupled to a L1 biosensor. See the supplemental Methods for specifics of liposome composition. The binding of LAIR-1-Ig (white bars) and CD300a-Ig (black bars) was analyzed by allowing the proteins to pass through the L1 sensor. The binding (resonance units or RU) for plateau values is shown. The RU are plotted after subtracting from the buffer control. Bar graph represents the average ± SEM. Results shown are from 8 independent experiments. (C) LAIR-1-Ig and CD300a-Ig were immobilized onto a CM5 sensor and a series of liposomes with different compositions (x-axis) were allowed to pass through the sensor and the binding analysis of liposomes to proteins was analyzed. See supplemental Methods for specifics of liposome composition. The binding (RU) for plateau values is shown. The RU are plotted after subtracting from the buffer control. Bar graph represents the average ± SEM. Results shown are from 2 independent experiments. (D) Liposomes of different compositions were incubated with CD300a-Ig fusion protein and allowed to associate. After 40 minutes of incubation the supernatants (S) and pellet (P) fractions were separated by ultracentrifugation and analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) analysis. The prestained protein markers (M) are also represented on the protein gel. Data shown are representative of 3 independent experiments.

This Feature Is Available To Subscribers Only