Figure 2. Initial crosslinking studies on in-cell relevance of α-hexamerization and a bicistronic reporter cassette for expression/rapid purification of hRNR (α₂)_m from mammalian cells.

(A) hRNR (α₂)_m expression platforms (Table S1A and S1B): Monocistronic cassette results in expression of untagged protein; Cassette A of DsRed and His₆-α; Cassette B of DsRed and eGFP-α. Merged confocal fluorescence images of live HeLa (1) and COS-1 (2) cells transfected with Cassette B. Scale bar in (1) and (2) each corresponds to 10 µm. (B) Protein content, fold-overexpression and activities in lysate (and of isolated, where applicable) of untagged-, His₆-and eGFP-α ectopically expressed in COS-1 cells, subsequent to transient transfection with either monocistronic cassette (Table S1A) or bicistronic reporter cassettes A or B (Table S1B). Corresponding values for the endogenous α were shown for comparison. Data, where applicable, are represented as mean +/− SD. Also see Figure S1A and S1B–C. (C) α-Hexamerization, subsequent to crosslinking in lysates, in response to ClF (50 nM) treatment of live COS-1 cells expressing untagged-α at 3.5 fold above the endogenous levels. Lanes a and b, results from BS3 (1 mM) treatment of the lysates (0.1 mg/ml) from ClF-treated (a) and untreated (b) cells. Lane c, recombinant His₆-α (0.15 µg/ml) treated in vitro with BS3 and subjected to identical sample preparation procedures as for lysates. Lane d, identical to lane c except additional presence of ClFTP (effectively, crosslinked α₆ standard - see Figure S1B–B for in vitro characterization). (D) SDS-PAGE of steps involved in His₆-α purification from COS-1 cells transfected with Cassette A. Lanes (left to right): lysate; supernatant after TALON^® incubation; wash 1; wash 2; elution; MW ladder. Arrows indicate His₆-α (92 kDa) and DsRed (27 kDa). Also see Figure S1D.