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. 2018 Jul 9;46(15):7886–7901. doi: 10.1093/nar/gky601

Figure 5.

Figure 5.

Over-stabilization of alt-P3 decreases active telomerase assembly in cells. (A) Activity of ZZF-tagged TERT RNP reconstituted in U20S cells with the AA48/54 set and Tetra-U/Tetra-G set of hTRmin template mutations. Cellular ZZF-TERT RNP was immunopurified (IP) and assayed directly on FLAG beads using template-specific primers in presence of all dNTPs. A background control expressed untagged TERT with WT (lane 10). Mutations are indicated with their corresponding ‘a-g’ states described for Figure 3. Values for rRAP* and rAct were calculated as described in Figure 3. (B) Immunoblot detection of ZZF-TERT in cell extracts and after immunopurification. Tubulin was used as the extract loading control for input samples and the FLAG-antibody light chain was non-specifically detected as a loading control for IP samples. (C) Northern blot detection of the overexpressed hTRmin in the cell extracts and after immunopurification. Normalized hTRmin RNA levels for the input and IP samples are shown relative to WT. (D–F) Experiments paralleling A–C for the AA49/55 set of hTRmin template mutations. Two background controls were included (lanes 8–9). Similar results were obtained from two independent experimental replicates.