Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: CD44 binds to F-HA, and the uptake in adherent RHAMM transfected cells is CD44-dependent . (A) F-HA probe uptake in adherent RHAMM-10T½ cells is significantly blocked. Values are the mean and SEM of n = 50 cells and asterisks indicate statistical significance ( p < 0.05) by function blocking anti-RHAMM antibodies but the effect is slight. (B) 240 kDa HA-sepharose pulls down CD44 standard, variant and soluble forms from 10T½ cell lysates, but in contrast, and as expected, HA 4 does not. (C) Anti-CD44 antibodies strongly block F-HA uptake in both parental and RHAMM-10T½ cells indicating that this receptor is primarily responsible for the RHAMM-mediated increase in F-HA internalization, and is the major endocytic HA receptor in adherent cells. Values are the mean and SEM of n = 50 cells. Asterisks indicate statistical significance ( p < 0.00001).

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Transfection, Blocking Assay, Variant Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: RHAMM affects CD44 expression . (A) Western blot analysis of adherent RHAMM-10T½ cell lysates reveals an approximately two-fold increase in the expression of CD44s protein compared to the parental cells (replicated twice). (B) Function blocking anti-RHAMM antibody reduces the display of CD44 on RHAMM-10T½ cells. (C) Reduction of CD44 display is observed when HA production by these cells is inhibited with 4MU.

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Expressing, Western Blot, Blocking Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: CD44 and RHAMM are displayed on suspended RHAMM-10T½ cells . (A) Single channel flow cytometry analysis of CD44 and RHAMM levels shows that CD44 levels are higher than RHAMM and that channel bleed through does not occur. (B) Multiplexed flow analysis of HA receptor display in RHAMM-10T½ cell subpopulations that bind low (blue, HA low bottom 5% of events) or high (red, HA high top 5% of events) amounts of F-HA probe show that CD44 is abundant in both subpopulations. In contrast, the highest RHAMM display is unique to the HA high subpopulation.

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: F-HA probe binding to suspended RHAMM-10T½ cells is reduced by anti-RHAMM but not anti-CD44 antibodies . (A,B) When compared to isotype matched non-immune IgG, function blocking anti-CD44 antibodies do not reduce binding of F-HA probe to suspended 10T½ cells (A) whereas anti-RHAMM antibody does (B) . (B, C) Expression of a dominant negative HA mutant that blocks HA binding to RHAMM significantly reduces F-HA internalization in attaching (2–12 h post subculture) but not firmly adherent 10T½ cells (24 h post subculture). Values are the mean and SEM of n = 50 cells. Asterisks indicate statistical significance p < 0.05.

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Binding Assay, Blocking Assay, Expressing, Dominant Negative Mutation, Mutagenesis