Figure S5.
Assessments of cytokine-independent proliferation induced by truncated his-GB1–tagged CRTDel52 mutants and secretory efficiencies of selected untagged CRTDel52 mutants. (A) Ba/F3-Mpl cells were electroporated with pcDNA-CoxG plasmids encoding N-terminal his-GB1–tagged full-length CRTWT, CRTDel52, CRTDel52Δ12, CRTDel52Δ19, CRTDel52Δ28, CRTDel52Δ36, or a control vector (Vec) and subsequently selected with Zeocin at 0.2 mg/ml. Left panel: Lysates from Ba/F3-Mpl cells expressing the indicated constructs were immunoblotted with anti-CRT(N) antibody (single analysis). Right panel: Cells were subsequently cultured in the absence of mouse IL-3, and proliferation was measured based on cell counting on the indicated days. Data are averaged from two independent proliferation experiments undertaken following a single electroporation of Ba/F3-Mpl cells. (B) IP of proteins from media and cell lysates of retrovirally transduced Ba/F3 cells expressing the indicated CRTDel52 constructs (those used for the analyses of Fig. 7). Media/cell ratios of CRTDel52 recovery were quantified and averaged across multiple experiments. Each data point in the graph represents an independent experiment. (C) Cell lysates from B were digested with PNGase F or EndoH as indicated before immunoblotting analyses. Data are representative of one to two independent experiments. MW, molecular weight.