American Society of Hematology

Figure 3.

$Figure 3. Purification of recombinant βig-h3 and identification of its secretion by imDCs. (A-C) Elution fractions of βig-h3 from the Ni-NTA-agarose column. 293T medium was mixed with Ni-NTA-agarose and bound βig-h3 was eluted using imidazole collecting 6 fractions (0.15 mL); 25 μL of each fraction was subjected to 10% SDS-PAGE under reducing conditions. The gels were either stained with Coomassie blue (A) or electroblotted. The blots were developed with either an anti–βig-h3 antibody (B) or an anti-myc antibody (C). (D) Purified βig-h3 and, as molecular weight standards, human IgM (Sigma-Aldrich) were subjected to 7.5% SDS-PAGE with (lanes 1-3) or without (lanes 4-6) 2-mercaptoethanol treatment. βIg-h3 was also purified in the presence (lanes 1 and 4) or absence (lanes 2 and 5) of iodoacetamide. As a control, purified human IgM was similarly analyzed (lanes 3 and 6). The gel was stained with Coomassie blue. (E) Media from 293T cells transfected with pβig-h3-MH (lane 1) or pcDNA3.1 (lane 2) or media from monocytes (lane 3), macrophages (lane 4), imDCs (lane 5), and mDCs (lane 6) were subjected to 10% SDS-PAGE, under reducing conditions, followed by Western blotting with a mouse anti–human βig-h3 antiserum. All molecular weight standards are in kilodaltons.$

Purification of recombinant βig-h3 and identification of its secretion by imDCs. (A-C) Elution fractions of βig-h3 from the Ni-NTA-agarose column. 293T medium was mixed with Ni-NTA-agarose and bound βig-h3 was eluted using imidazole collecting 6 fractions (0.15 mL); 25 μL of each fraction was subjected to 10% SDS-PAGE under reducing conditions. The gels were either stained with Coomassie blue (A) or electroblotted. The blots were developed with either an anti–βig-h3 antibody (B) or an anti-myc antibody (C). (D) Purified βig-h3 and, as molecular weight standards, human IgM (Sigma-Aldrich) were subjected to 7.5% SDS-PAGE with (lanes 1-3) or without (lanes 4-6) 2-mercaptoethanol treatment. βIg-h3 was also purified in the presence (lanes 1 and 4) or absence (lanes 2 and 5) of iodoacetamide. As a control, purified human IgM was similarly analyzed (lanes 3 and 6). The gel was stained with Coomassie blue. (E) Media from 293T cells transfected with pβig-h3-MH (lane 1) or pcDNA3.1 (lane 2) or media from monocytes (lane 3), macrophages (lane 4), imDCs (lane 5), and mDCs (lane 6) were subjected to 10% SDS-PAGE, under reducing conditions, followed by Western blotting with a mouse anti–human βig-h3 antiserum. All molecular weight standards are in kilodaltons.

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