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. Author manuscript; available in PMC: 2014 Nov 21.
Published in final edited form as: Cell. 2013 Nov 21;155(5):1034–1048. doi: 10.1016/j.cell.2013.10.021

Figure 3. Characterization of highly purified L1 RNPs.

Figure 3

(A) L1 interactors characterized by MALDI-TOF-PMF MS. Ctrl – empty vector-containing cells. (B) Relative LEAP activity from affinity purified RNPs and RNPs made by sucrose cushion velocity sedimentation. Vector, RNP purified from cells transfected with empty pCEP puro vector; Boiled, RNP sample was boiled for 10 min at 100°C before the reaction. Data are represented as mean +/− SEM. (C) Specific LEAP activity normalized to total L1 mRNA in each sample. LEAP activity of RNP prepared from sucrose cushion was assigned as 1. Please see Figures S3 for calculations. Data are represented as mean +/− SEM. (D) Tandem affinity purification of L1 RNPs. L1 RNPs were first purified from ORF2p (3xFlag) and then ORF1p. Sypro Ruby stained supernatant (super) and elution from triplicate ORF1p purifications are shown for both ORFeus-Hs and L1RP; ORF1p and ORF2p levels were quantified against a BSA standard (Figure S4). (E) Indirect immunofluorescent imaging of ORF1p and ORF2p in HeLa and HEK293T cells. Ratio calculation for HeLa and HEK293T cells expressing ORF1p alone or both ORF1p and ORF2p is shown. Numbers in the table are counts of cells expressing ORF1p alone or both ORFs above background. Please see Figures S5 for more imaging and controls.