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. 2008 Feb 25;180(4):705–712. doi: 10.1083/jcb.200709100

Figure 2.

Figure 2.

Screening of the cDNA-YFP1 library for ERGIC-53 interaction partners. (A) COS-1 cells were transfected with the indicated bait and prey constructs and analyzed by FACS as described in Materials and methods. Coexpression of YFP2–ERGIC-53 and the cDNA-YFP1 library resulted in the specific detection of 1.63% YFP positive (YFP+) cells. In a nonsaturating screen, ∼500 fluorescent cells were collected by FACS and total DNA was extracted and transformed into bacteria, which resulted in the recovery of several hundred prey clones. (B) 48 prey clones were randomly selected and plasmids were isolated and individually assayed by YFP PCA with YFP2–ERGIC-53 in COS-1 cells. Fluorometric analysis revealed that prey plasmids 17, 32, 33, and 44 (indicted by red boxes) were positive and reconstitute fluorescent YFP when expressed with YFP2–ERGIC-53. Bars represent fluorometric values of a single screening experiment. The threshold for a positive hit was set to 250 arbitrary fluorescence units, which corresponds to a ∼1.5-fold induction in YFP fluorescence in comparison to untransfected cells. (C) Sequence analyses identified α1-AT as a cDNA insert in all four positive prey plasmids.