American Society of Hematology

Figure 2.

$Figure 2. Generation of the stable cell lines expressing the lipid raft–anchored forms of Cbl-b. (A) Schematic diagram of the mutant forms of Cbl-b used in the current experiments. Both GEM-Cbl-b and GEM-Cbl-b (C373A) are chimeric molecules with the myristoylation and palmitoylation signals from c-Src (AA 1-16), in which Ser3 was substituted to Cys (GEM-tag). These cDNAs were subcloned into the pSVL expression vector. 4H indicates 4-helix; EF, EF-hand Ca++ binding domain; RING, RING finger; Pro rich, Pro-rich region; LZ, leucine zipper domain. (B) RBL-2H3 cells were stably cotransfected with pSVL-GEM-Cbl-b or pSVL-GEM-Cbl-b (C373A) together with pSV2-neo vector and selected with G418. Two or 3 positive cloned lines overexpressing each of the different kinds of chimeric proteins were selected for further analysis. Total cell lysates from the parental RBL-2H3 and selected cloned lines were analyzed by the immunoblotting with anti-Cbl-b, anti-HA, and anti-FcϵRIβ antibodies as an internal control. (C) GEM-Cbl-b mainly localizes in GEMs. Cell homogenates from control RBL-2H3 cells and cells overexpressing GEM-Cbl-b were fractionated by the sucrose density gradient centrifugation, and fractions 1 to 3, 4 to 6, and 7 to 10 were each collected and reacted with anti-Cbl-b antibody prebound to protein-G-Sepharose, respectively. The immunoprecipitates from collected fractions were analyzed by the immunoblotting with anti-HA mAb. Similar results were obtained when the other cloned lines were examined. The results were representative of 4 experiments.$

Generation of the stable cell lines expressing the lipid raft–anchored forms of Cbl-b. (A) Schematic diagram of the mutant forms of Cbl-b used in the current experiments. Both GEM-Cbl-b and GEM-Cbl-b (C373A) are chimeric molecules with the myristoylation and palmitoylation signals from c-Src (AA 1-16), in which Ser3 was substituted to Cys (GEM-tag). These cDNAs were subcloned into the pSVL expression vector. 4H indicates 4-helix; EF, EF-hand Ca⁺⁺ binding domain; RING, RING finger; Pro rich, Pro-rich region; LZ, leucine zipper domain. (B) RBL-2H3 cells were stably cotransfected with pSVL-GEM-Cbl-b or pSVL-GEM-Cbl-b (C373A) together with pSV2-neo vector and selected with G418. Two or 3 positive cloned lines overexpressing each of the different kinds of chimeric proteins were selected for further analysis. Total cell lysates from the parental RBL-2H3 and selected cloned lines were analyzed by the immunoblotting with anti-Cbl-b, anti-HA, and anti-FcϵRIβ antibodies as an internal control. (C) GEM-Cbl-b mainly localizes in GEMs. Cell homogenates from control RBL-2H3 cells and cells overexpressing GEM-Cbl-b were fractionated by the sucrose density gradient centrifugation, and fractions 1 to 3, 4 to 6, and 7 to 10 were each collected and reacted with anti-Cbl-b antibody prebound to protein-G-Sepharose, respectively. The immunoprecipitates from collected fractions were analyzed by the immunoblotting with anti-HA mAb. Similar results were obtained when the other cloned lines were examined. The results were representative of 4 experiments.

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