1. by Subburaj Ilangumaran, Anne Briol, and Daniel C. Hoessli
CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes
by Subburaj Ilangumaran, Anne Briol, and Daniel C. Hoessli
Blood
Volume 91(10):
May 15, 1998
©1998 by American Society of Hematology

2. Association of CD44 with low-density plasma membrane fractions in human T lymphocytes.
Association of CD44 with low-density plasma membrane fractions in human T lymphocytes. One hundred million human PBMC (left panel) or PHA blasts (right panel) were extracted in TKM buffer containing 0.5% TX-100, and the lysates were subjected to equilibrium gradient centrifugation as described in the Materials and Methods. Eleven 1-mL fractions were collected from the top, and 20 μL of each fraction (2 through 11) was electrophoresed under nonreducing (CD44, CD45, MHC-I, and CD59) or reducing (Lck and Fyn) conditions. Separated proteins were detected by ECL-based Western blot. Fractions 3 through 6 correspond to the 5% to 36% sucrose interface. Data shown are representative of at least three experiments. Identical distribution profiles were obtained when 0.5% Brij-58 was used for extraction. Fraction 1 did not contain any of the cell surface or intracellular proteins tested.
Subburaj Ilangumaran et al. Blood 1998;91:
©1998 by American Society of Hematology

3. Distribution of the glycosphingolipid GM1 in equilibrium sedimentation gradients.
Distribution of the glycosphingolipid GM1 in equilibrium sedimentation gradients. In (A), 10 μL of the gradient fractions shown in Fig 1 was dot-blotted onto nitrocellulose filters, blocked, and probed with HRP-conjugated cholera toxin followed by ECL detection. The total protein profile of the gradient fractions of TX-100 lysate of PBMC is shown in (B). A similar profile obtained for PHA blasts is not shown.
Subburaj Ilangumaran et al. Blood 1998;91:
©1998 by American Society of Hematology

4. (A) Endothelial cell CD44 partitions into caveolin containing low-density membrane fractions.
(A) Endothelial cell CD44 partitions into caveolin containing low-density membrane fractions. ECV304 cells grown in 10-cm Petri dishes were extracted in TKM-0.5% TX-100 (upper panel) or 60 mmol/L OTG (lower panel) at 4°C for 30 minutes. The lysates were subjected to equilibrium sedimentation, and the fractions were tested for the distribution of CD44, CD59, or caveolin as described in Fig 1. Caveolin was analyzed under reducing conditions. (B) CD44 and CD59 do not interact directly in the low-density membrane fractions. Surface biotinylated ECV304 cells were extracted with TKM-0.5% TX-100 and ultracentrifuged in sucrose gradients, and the GPI-rich top fractions 3 through 6 were pooled. One milliliter of the pool was precleared with Pansorbin and immunoprecipitated with protein A/G beads coated with antibodies against CD59 or CD44. SDS-PAGE separated proteins were detected by Western blot using streptavidin-HRP.
Subburaj Ilangumaran et al. Blood 1998;91:
©1998 by American Society of Hematology

5. Stimulation of protein tyrosine phosphorylation via CD44.
Stimulation of protein tyrosine phosphorylation via CD44. Freshly isolated human PBMC were stimulated by immobilized anti-CD44 MoAb as described in the Materials and Methods. At the indicated time points, cells were lysed and equivalent amounts of lysates were electrophoresed under reducing conditions, blotted, and probed for tyrosine phosphorylated proteins by PY20 followed by GAM-AP and ECL detection (upper panel). Lane 1, unstimulated cells; lanes 2 through 6, cells plated in CD44 MoAb-coated wells, lane 7, cells plated in control mouse IgG-coated wells (C). Equivalent protein loading in the wells was verified by Ponceau-S staining of the blots, as well as by probing for Lck and Fyn kinases (lower panels).
Subburaj Ilangumaran et al. Blood 1998;91:
©1998 by American Society of Hematology

6. Kinase activities associated with CD44 are localized in low-density plasma membrane domains.
Kinase activities associated with CD44 are localized in low-density plasma membrane domains. (A) One milliliter of total Brij-58 lysate from 10 ×106 PBMC was precleared with Pansorbin and immunoprecipitated on protein A/G beads coated with indicated antibodies or a control MoAb (against hapten TNP) and blocked subsequently with FCS. After washing, the beads were assayed for the associated kinase activity at 30°C for 15 minutes. The reaction was stopped by boiling in sample buffer, and the phosphorylated proteins separated by SDS-PAGE were detected by autoradiography. (B) After gradient centrifugation of the Brij-58 extract as in Fig 1, the top (3 through 5) and bottom (9 through 11) fractions were pooled separately. The bottom pool containing 80% of the cellular proteins was diluted four times to equalize the protein concentration. Immunoprecipitation with indicated antibodies from the pooled top or bottom fractions and kinase reaction were performed as in (A). The autoradiograms were obtained after overnight exposure at room temperature. Similar results were obtained with PHA blasts (not shown). Kinase activities associated with CD44 are better preserved in whole lysates or gradient top fractions when Brij-58 was used; experiments with TX-100–lysed samples required a much longer exposure time, but showed an identical phosphoprotein profile (not shown).
Subburaj Ilangumaran et al. Blood 1998;91:
©1998 by American Society of Hematology

7. CD44 is associated with Lck and Fyn kinases in the low-density membrane domains.
CD44 is associated with Lck and Fyn kinases in the low-density membrane domains. After kinase assay on CD44 immunoprecipitates from pooled low-density fractions as in Fig 5B, left panel top, the immune complexes were washed in 0.5% Brij-58 lysis buffer, dissociated by SDS, and reimmunoprecipitated using the indicated antibodies. The immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography after 4 days of exposure. The total phosphoprotein profile associated with the primary CD44 immunoprecipitate is shown in the first lane.
Subburaj Ilangumaran et al. Blood 1998;91:
©1998 by American Society of Hematology