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. 2019 Jul 1;116(29):14620–14629. doi: 10.1073/pnas.1903432116

Fig. 4.

Fig. 4.

EMS cooperates with RALY to promote E2F1 mRNA stability. (A) A549 cells were infected with lentiviruses expressing either control shRNA (shctrl) or EMS shRNA (shEMS). Forty-eight hours later, cells were incubated with actinomycin D (2 μg/mL) for the indicated periods of time. Total RNA was then analyzed by real-time RT-PCR to examine E2F1 mRNA stability. Data shown are mean ± SD (n = 3). (B) A549 cells were infected with lentiviruses expressing pSin-control or pSin-Flag-RALY. Forty-eight hours later, cell lysates were immunoprecipitated with anti-Flag antibody. RNAs in immunoprecipitates (IP) were then analyzed by real-time RT-PCR to examine EMS and E2F1 mRNA levels. Data shown are mean ± SD (n = 3). **P < 0.01. The input and immunoprecipitates were also analyzed by Western blotting. (C) Lysates from A549 cells were incubated with either sense or antisense biotin-labeled DNA oligomers corresponding to EMS, followed by the pull-down experiments using streptavidin-coated beads. The pull-down complexes were analyzed by Western blotting. (D) In vitro-synthesized EMS or its antisense RNA was incubated with purified recombinant Flag-RALY bound with M2 beads. The bead-bound RNAs were then eluted as templates for RT-PCR analysis. (E) A549 cells were infected with lentiviruses expressing control, EMS shRNA, or both EMS shRNA and RALY. Forty-eight hours later, cells were incubated with actinomycin D (2 μg/mL) for the indicated periods of time. Total RNA was then analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3). (F) A549 cells were infected with lentiviruses expressing control (ctrl), EMS, RALY shRNA (shRALY), or both EMS and RALY shRNA. Forty-eight hours later, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3). *P < 0.05; **P < 0.01, ns., no significance. (G) A549 cells were infected with lentiviruses expressing ctrl, EMS, RALY shRNA, or both EMS and RALY shRNA. Forty-eight hours later, cell lysates were analyzed by Western blotting. (H) A549 cells were infected with lentiviruses expressing control, EMS shRNA, RALY, or both EMS shRNA and RALY. Forty-eight hours later, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (I) A549 cells were infected with lentiviruses expressing control, EMS shRNA, RALY, or both EMS shRNA and RALY. Forty-eight hours later, cell lysates were analyzed by Western blotting. (J) A549 cells transduced with lentiviruses expressing either control shRNA or EMS shRNA were transfected with or without Flag-RALY as indicated. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. RNAs present in immunoprecipitates (IP) were then analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3). *P < 0.05. The input and immunoprecipitates were also analyzed by Western blotting. (K) Purified recombinant Flag-RALY bound with M2 beads was incubated with in vitro-synthesized E2F1 3′-UTR, EMS, and its antisense RNA in the indicated combination. The bead-bound RNAs were then eluted as templates for RT-PCR analysis. (L) In vitro-synthesized biotin-labeled E2F1 3′-UTR and unlabeled EMS plus Flag-RALY were incubated at room temperature for 30 min. The mixtures were first immunoprecipitated with Flag-M2 beads, followed by the elution step with 3 × FLAG peptides. Half of the eluent was subjected to Western blot and RT-PCR analysis. The rest of the eluent was further immunoprecipitated with streptavidin beads. After extensive washing, the immunoprecipitates were analyzed by Western blotting and RT-PCR.