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Blood, Vol. 113, Issue 3, 575-584, January 15, 2009
Dexamethasone augments CXCR4-mediated signaling in resting human T cells via the activation of the Src kinase Lck
Blood Ghosh et al.
113: 575
Supplemental materials for: Ghosh et al
Files in this Data Supplement:
- Figure S1 (JPG, 59.1 KB)
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(A) Kinetic of DM-mediated Lck phosphorylation. Jurkat E6.1 T cell was treated with 1uM of DM for different time like 30 seconds, 1, 5, 15, and 30 minutes. Rest of the procedure to immunoblot the phospho Lck and densitometry to present data was mentioned as before. Similar kinetics were observed using primary cells. (B) Site-specific phosphorylation on the Lck molecule upon DM and/or CXCL12 treatment. Resting and anti-CD3 mAb-activated T cells were treated with DM, CXCL12 or the combination for 5 minutes after which total Lck was immunoprecipitated and site specific phosphorylation on the tyrosine residue was examined using the antibody Y394 (Cell Signal, Danvers, MA) and Y505 (Cell Signal, Danvers, MA). Phosphoserine levels were also examined in these assays. Total Lck was also immunoblotted. The results were presented as normalized value as fold change over control. The number of donors examined in these studies was 2.
- Figure S2 (JPG, 52.1 KB)
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(A) DM and CXCL12 induced activation of Lck kinase activity in treated human T cells. Resting human T cells were treated with DM, CXCL12 or the combination after which the sample was examined using an Lck kinase assay kit (Cell Signal, Danvers, MA). The preparation of the cell lysates and kinase activity from these lysates was performed according to the manufacturer’s instructions. Kinase activity was expressed as fold change over the control (+/− SE). * indicate the value is significant at p<0.05 level, while ** indicate a significance value at p<0.01. The number of donors examined in these assays was 3. (B) Dissociation of Lck from the CD4 molecule in response to DM and/or CXCL12 treatment. Human T cells and Jurkat cells were treated with CXCL12 (100ng/ml) and DM (1 µM) or the combination for 5 minutes and then lysed with RIPA buffer as described in Fig 5A. Total CD4 was immunoprecipitated using the anti-CD4 antibody and Lck was immunoblotted from the immunoprecipitated CD4. Total amount CD4 was also immunoblotted as loading control. The value of densitometry was normalized as ratio of Lck over CD4 and then presented as fold changed over control. The number of donors examined in these assays was 2.
- Figure S3. Colocalization of GR, Lck and CD45 upon treatment of DM and/or CXCL12 (JPG, 52.8 KB)
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Primary T cells were treated with DM, CXCL12 or the combination for 5 minutes as described in the previous figures. The cells were then fixed with 4% paraformaldehyde followed by blocking with 10% FCS for 1 h. Primary antibody against GR (anti-mouse, abCAM, Cambridge, MA), Lck (anti-rabbit, Cell Signal, Danvers, MA) and CD45 (anti-goat, Santa Cruz Technology, Santa Cruz, CA) from different host were used at 1:100 for 1 h at 37°C. Secondary antibody labeled with alexfluor-647 (blue), -488 (green) and -555 (red) targeted to primary antibody against GR, Lck, and CD45 was used for 1 h at 1:100 at 37°C. Images were acquired by a Carl Zeiss, LSM 510 (Thornwood, NY). Image of different color was colocalized and development of white color on the image was indicted by arrow (violet). Single plane images were examined here. Twenty five cells were counted per different fields and the numbers of cells demonstrating a colocalized phenotype (pink-white cells) were expressed as Percent Colocalization (+/− SE). The number of donors examined was 4.
- Figure S4 (JPG, 54.5 KB)
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(A) No interaction between CXCR4 and Lck upon DM and/or CXCL12 treatment. Resting T cells were treated with DM, CXCL12 or the combination after which lysates were prepared with RIPA buffer and protease inhibitor. Antibody for CXCR4 (abCAM, Cambridge, MA) and Lck (Cell Signal, Danvers, MA) were utilized to immunoprecipitate total CXCR4 and Lck followed by Western blotting with the reciprocal antibodies. The total immunoprecipitation of each molecule was also confirmed by Western blot analysis. The number of donors examined in these assays was 2. (B) DM but not CXCL12 induces the phosphorylation of Lck in a TCR-negative cell line. J E6.1 and JRT3.1 (a clone of J E6.1 which is devoid of beta-chain in TCR) were treated with DM, CXCL12 or the combination for 5 minutes after which total Lck was immunoprecipitated and the phosphorylation status was examined by Western blotting. Densitometry of each band was performed, and the data was normalized against the total Lck. The final data is presented as fold change over control.
- Figure S5 (JPG, 54 KB)
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(A) DM treatment of T cells does not influence the cell surface expression of CXCR4 at 15 minutes or 2 hours. Primary human T cells and Jurkat cells were treated with CXCL12 (100ng/ml), DM (1 µM) or the combination for 15 minutes (a) and 2 hours (b). The cells were subsequently fixed with 2% paraformaldehyde solution for 1 hour followed by incubation with anti-CXCR4 antibody labeled with FITC. Data acquisition was performed by forward scatter versus side scatter. “MFI” indicates the mean fluorescence intensity. (B) DM and CXCL12 do not influence CD48 and CD45 association with lipid rafts. Resting human T cells were treated with CXCL12 (100ng/ml) and DM (1 µM) or the combination for 5 minutes. The cell membranes were fractionated as previously described.22,23 The CD48 and CD45, two marker proteins of raft and non-raft, respectively, were immunoblotted from the nine fractions in each preparation. No alterations in the localization were also observed for CD4, CD45, CD48 or CXCR4 upon treatment with DM, CXCL12 or the combination (CD4 and CXCR4, not shown). The number of donors examined in these assays was 3.
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