Figure 4
Figure 4. CXCR4-dependent chemotaxis is inhibited by p52Shc mutants lacking YY239/240 or Y317. (A) Migration of the Jurkat T-cell transfectants expressing HA-tagged p52Shc or the Shc1F/Shc2F mutants (left), or of PBLs from 3 different donors transiently transfected with empty vector (ctr) or HA-tagged p52Shc or the Shc1F/Shc2F mutants (right), measured after treatment for 2 hours with SDF-1α. The data are presented as relative migration, with the migration index of SDF-1α–treated control cells (transfected with empty vector) taken as 100% (n ≥ 3). PBLs were cotransfected with a GFP reporter, and migration was measured on GFP+ cells. Transfection efficiencies were consistently approximately 50%. The relative amount of HA-Shc (wild-type or mutant) compared with endogenous p52Shc in transiently transfected PBLs was 40% to 50%, as detected by laser densitometric analysis of anti-Shc immunoblots. (B, left) Flow cytometric analysis of CXCR4 surface expression on Shc-deficient JSL1 cells stably transfected with either empty vector (vect) or expression constructs encoding either HA-tagged p52Shc (p52), or p46Shc (p46), or both isoforms (p52/p46). Cells transfected with the latter construct frequently harbor, in addition to p52Shc and p46Shc, an immunoreactive band with a slightly higher electrophoretic mobility than p52Shc, which may result from usage of a 36-bp upstream in-frame ATG in the genomic locus when the p66Shc initiator ATG is mutated. An anti-Shc immunoblot of the respective cell lysates is shown above. Equal amounts of lysates were loaded, as evaluated by reprobing the filter with antiactin mAb (not shown). (B, right) Migration of Jurkat cells and of the JSL1 cell transfectants, measured after 2-hour treatment with SDF-1α (n ≥ 3). (C) Migration of Jurkat cells and JCaM1 cells, measured after 2-hour treatment with SDF-1α. (D) Migration of Jurkat cells, measured after 2-hour treatment with SDF-1α in the presence or absence of PTX (500 ng/mL) or AG490 (100 μM) or after Jak2 knockdown by siRNA (n ≥ 3). Data in panels B-D are presented as relative migration, with the migration index of SDF-1α–treated Jurkat cells taken as 100%. Error bars indicate SD.

CXCR4-dependent chemotaxis is inhibited by p52Shc mutants lacking YY239/240 or Y317. (A) Migration of the Jurkat T-cell transfectants expressing HA-tagged p52Shc or the Shc1F/Shc2F mutants (left), or of PBLs from 3 different donors transiently transfected with empty vector (ctr) or HA-tagged p52Shc or the Shc1F/Shc2F mutants (right), measured after treatment for 2 hours with SDF-1α. The data are presented as relative migration, with the migration index of SDF-1α–treated control cells (transfected with empty vector) taken as 100% (n ≥ 3). PBLs were cotransfected with a GFP reporter, and migration was measured on GFP+ cells. Transfection efficiencies were consistently approximately 50%. The relative amount of HA-Shc (wild-type or mutant) compared with endogenous p52Shc in transiently transfected PBLs was 40% to 50%, as detected by laser densitometric analysis of anti-Shc immunoblots. (B, left) Flow cytometric analysis of CXCR4 surface expression on Shc-deficient JSL1 cells stably transfected with either empty vector (vect) or expression constructs encoding either HA-tagged p52Shc (p52), or p46Shc (p46), or both isoforms (p52/p46). Cells transfected with the latter construct frequently harbor, in addition to p52Shc and p46Shc, an immunoreactive band with a slightly higher electrophoretic mobility than p52Shc, which may result from usage of a 36-bp upstream in-frame ATG in the genomic locus when the p66Shc initiator ATG is mutated. An anti-Shc immunoblot of the respective cell lysates is shown above. Equal amounts of lysates were loaded, as evaluated by reprobing the filter with antiactin mAb (not shown). (B, right) Migration of Jurkat cells and of the JSL1 cell transfectants, measured after 2-hour treatment with SDF-1α (n ≥ 3). (C) Migration of Jurkat cells and JCaM1 cells, measured after 2-hour treatment with SDF-1α. (D) Migration of Jurkat cells, measured after 2-hour treatment with SDF-1α in the presence or absence of PTX (500 ng/mL) or AG490 (100 μM) or after Jak2 knockdown by siRNA (n ≥ 3). Data in panels B-D are presented as relative migration, with the migration index of SDF-1α–treated Jurkat cells taken as 100%. Error bars indicate SD.

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