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1.
Figure 2

Figure 2. Optimal CP of SIINFEKL requires substrates with N-terminal extensions longer than 35 AA. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Representative results of CP and DP for the indicated constructs. Data corresponds to the average of two wells and is expressed as the % of β-gal activity (counts/second) of the maximal signal in the experiment.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
2.
Figure 1

Figure 1. CP but not DP of SIINFEKL requires flanking sequences at both ends of the determinant. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Representative results of CP and DP for the indicated constructs. Data corresponds to the average of two wells and is expressed as the % of β-gal activity (counts/second) of the maximal signal in the experiment.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
3.
Figure 5

Figure 5. Changes in the C-terminal extension do not affect DP presentation by Mϕ. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Representative DP by Mϕ infected with 10 PFU VACV expressing the indicated constructs. Data corresponds to the average of three wells and is expressed as the % of β-gal activity (counts/second) of the maximal signal in the experiment.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
4.
Figure 3

Figure 3. Optimal CP of SIINFEKL requires C-terminal extensions longer than 23 AA. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Representative results of CP (A) and DP (B) for the indicated constructs. Data corresponds to the average of two wells and is expressed as the % of β-gal activity (counts/second) of the maximal signal in the experiment.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
5.
Figure 6

Figure 6. Variable effects on CP of SIINFEKL as a result of changes in the sequence of the N- and/or C- terminal extensions. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Representative results of CP and DP for the indicated constructs. Data corresponds to the average of two wells and is expressed as the % of β-gal activity (counts/second) of the maximal signal in the experiment.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
6.
Figure 4

Figure 4. Inhibition of the donor cell proteasome blocks CP of the smallest cross-presented construct. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Representative CP results for the indicated constructs expressed by cells treated or not for two hwith 2 µM lactacystin and washed before placing them in the CP assay. Data corresponds to the average of two wells and is expressed as the % of β-gal activity (counts/second) of the maximal signal in the experiment.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
7.
Figure 8

Figure 8. The rules of in vitro CP and in vivo cross-priming are similar. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

C57BL/6 mice were inoculated with CFSE labeled OT1-Ty1.1 cells and immunized in the flank with A9-T7 that had been infected with VACV and transfected with the indicated constructs. Four days later, the proliferation of OT1-Ty1.1 cells in the draining inguinal lymph node was analyzed by FACS. The data shows CFSE fluorescence in gated Thy1.1+, CD8+ cells for individual mice. A total of 106 cells in the lymphocyte gate where acquired in all cases.

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.
8.
Figure 7

Figure 7. The reduced CP of 46-SIINFEKL-21 is not due to a shorter half-life. From: The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation.

Plasmids coding for the indicated constructs driven by the T7 promoter were transfected into A9 cells and protein expression was induced by infection with a vaccinia virus expressing the T7 polymerase (VACV-T7). Sixteen h after infection the cells were pulsed with 35S M and C for 10 min and chased with cold M and C for the indicated times. The samples for 46-SIINFEKL-21 (8.63 Kd) and 46-SIINFEKL-22 (8.76 Kd) and those of 46-SIINFEKL-25 (9.15 Kd) and empty vector are two different gels run, transferred to nitrocellulose paper, and exposed at the same time. The arrow indicates the approximate position expected for the three constructs. As expected, when electrophoresed in the same gel 46-SIINFEKL-25 runs somewhat slower than the other two constructs (not shown). In addition to their absence in the empty vector control, the ~9 kD bands were absent in cells transfected with 46-SIINFEKL-121 (which expressed a long-lived ~19 kD band, not shown).

Xueying Ma, et al. J Immunol. ;182(8):4601-4607.

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