Fig 1: RPLP1/2 knockdown increases the rates of DENV structural protein synthesis and turnover. HeLa cells expressing HA-C-prM-E?208-FLAG were transfected with control or RPLP1/2 siRNAs and then metabolically pulse-labeled with 35S-methionine/cysteine for 30 min followed by chase with cold methionine/cysteine. Cells were harvested immediately after the pulse, 3 or 6 hours after the chase for lysis and IP with a-HA and a-FLAG antibodies. (A) An autoradiogram of triplicate IP samples for HA-C-prM and E?208-FLAG is shown for the pulse (0 h) and chase samples. Controls in the two leftmost lanes were not induced with tetracycline. (B) The left panel shows raw quantification of band intensities and the right panel shows levels normalized to the 0 h time point for HA-C-prM. (C) Same as in (B) except data show E?208-FLG. A representative experimenti is shown and its quantifications were done by averaging the measurements of triplicate bands in the autoradiagram.
Fig 2: Deletion of transmembrane domain (TMDs) within the prM protein abrogates the effect of RPLP1/2 knockdown on viral protein expression. (A) Tetracycline inducible HeLa cells expressing DENV structural proteins were transfected with control or RPLP1/2 siRNAs and levels of HA-C-prM and E?208-FLAG were analyzed by western blot using the respective tag antibodies. Analysis of cells expressing variants lacking TMD3 (B) or both TMD 2 and 3 (C) is shown. Representative experiments and shown and their quantifications are shown on the right. The graphs show mean values ± SD. Asterisks indicate p values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 3: RPLP1/2 depletion leads to ribosome pausing after TMDs on cellular mRNAs. (A–F) Examples of cellular genes encoding multiple TMDs which show an increase in the ribosome pausing downstream of the TMDs when RPLP1/2 are depleted.
Fig 4: Kaplan-Meier survival curves for high versus low RPLP1 expression in 81 patients with TNBC. Patients in the high expression group had significantly shorter overall survival than those in the low expression group
Fig 5: RPLP1 expression in TNBC and normal breast cells. a RPLP1 protein expression was higher in TNBC cells than in the normal breast cell line. The bar chart demonstrates the ratio of RPLP1 protein to ß-actin using quantitative analysis. b RPLP1 mRNA expression was also higher in TNBC cells than in the normal breast cell line. Data are mean ± SD for three independent experiments. *P < 0.05, compared to the MCF-10A cell line
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