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. 2021 Apr 28;220(7):e202009179. doi: 10.1083/jcb.202009179

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© 2021 Venkatesan et al.

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Figure S3. — Secreted forms of mutant CRT multimerize, and CRT_Ins forms disulfide-linked multimers similar to CRT_Del52. (A–C) HEK293T cells were transiently transfected with plasmids encoding full-length his-GB1–tagged CRT_Del52 (A) or untagged CRT_WT, CRT_Ins5, CRT_Ins5-2CA [CRT_{Ins5(C419A/C423A)}], CRT_Ins5-3CA [CRT_{Ins5(C163A/C419A/C423A)}], or plasmid lacking CRT (Vec; B and C). Cell media from untransfected (HEK) or CRT_Del52-transfected cells were purified on a nickel column and the eluates analyzed by immunoblot following SDS-PAGE under reducing or nonreducing conditions. (B and C) Lysates from indicated cells were separated by SDS-PAGE under reducing (8% gels) or nonreducing (4–20% gradient gels) conditions and immunoblotted with indicated antibodies. Data are representative of two (A) and three (B and C) sets of analyses. In blots following nonreducing SDS-PAGE, species consistent with the size of CRT monomers, dimers, and endogenous CRT are indicated. MW, molecular weight.