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Blood, Vol. 110, Issue 13, 4331-4340, December 15, 2007
Identification of CMS as a cytosolic adaptor of the human pT chain involved in pre-TCR function
Blood Navarro et al.
110: 4331
Supplemental materials for: Toribio et al
Files in this Data Supplement:
- Table S1. Primer list (PDF, 35KB)
- Figure S1. CMS/actin-rich endocytic vesicles are selectively formed in pre-T cells (PDF, 726 KB) -
Primary pre-TCR+ thymocytes isolated ex-vivo (A) and mature T (Jurkat) and pre-T (SupT1) cell lines transfected with CMS-GFP (B) were labelled with Phalloidin-TRITC and analyzed by confocal microscopy for the presence of actin-rich vesicles.
- Figure S2. Sorting of pTα to the lysosomal compartment is dependent on an intact actin cytoskeleton (PDF, 79 KB) -
SupT1 cells either treated with Cytochalasin D (10µM, Calbiochem) for 1h, or left untreated, were incubated with an anti-pT mAb for 15 min at 37°C, washed, and either fixed before staining, or incubated for additional 15 min (30min) prior to fixation. Internalized pT was detected after fixation with FITC-coupled anti-IgG2a plus Alexa488-anti-FITC, and co-localization of pT to the endosomal or lysosomal compartments was assessed by confocal microscopy after labelling with anti-EEA1 or anti-Lamp1 plus Alexa555-anti-IgG1, respectively. The mean percentage of co-localization ± SEM (n=20-30 cells) is shown. p values were calculated using a paired t test. *p<0.0001.
- Figure S3. Neither CMS nor polymerized actin co-localize with the conventional CD3-TCRα β complex internalized upon receptor engagement on mature T cells (PDF, 167 KB) -
Jurkat cells transiently transfected with CMS-GFP were incubated with an anti-CD3 mAb for 15 (A) or 30 min. (B) at 37ºC, washed, and fixed. After fixation, internalized CD3 was detected by labelling with Alexa555-coupled anti-mouse IgGs. Co-localization of internalized CD3 to actin-rich endocytic vesicles was assessed by confocal microscopy after labelling with Alexa647-coupled Phalloidin.
- Figure S4. Ca2+ mobilization in JRpTα WT cells is not affected by CMS over-expression (PDF, 87 KB) -
JRpTWT cells either transiently transfected with either full length CMS or an empty vector were loaded with Indo-1 AM. Washed cells were analyzed for 510 (FL4 channel) and 400 (FL5 channel) nm emissions at different time points both before and after stimulation with anti-CD3. Ionophore treatment was used to control for cell viability and intact calcium stores. Data are represented as the FL4/FL5 emission ratio analyzed by flow cytometry using the Three Star FlowJo software (BD Biosciences).
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