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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Pigment Cell Melanoma Res. 2014 Jun 16;27(5):801–812. doi: 10.1111/pcmr.12259

Figure 5. siRNA mediated RPL13 knockdown caused protein synthesis inhibition, and effects seen were not due to an effect on general protein synthesis inhibition.

Figure 5

(A, B) Cells transfected with RPL siRNA were subjected to methionine starvation then azidohomoalanine (AHA) incubation before collecting lysates. Biotin was attached to the AHA in newly synthesized proteins before performing western blotting and probing membranes with streptavidin-HRP to identify the extent of new protein synthesis in UACC 903 (A) or the effect of siRPL13 inhibition in UACC 903 and 1205 Lu (B). Erk2 served as the control for equal protein loading. (C) UACC 903 cells were treated with varying concentrations of the protein synthesis inhibitors cycloheximide, puromycin, and anisomycin for 4 hours before subjecting cells to methionine starvation then AHA incubation prior to collecting lysates. Biotin was attached to the AHA in newly synthesized proteins before performing western blotting and probing the membrane with streptavidin-HRP to identify the extent of new protein synthesis. Erk2 was used as a control for equal protein loading. (D) UACC 903 cells were treated with varying concentrations of cycloheximide, puromycin or anisomycin for 24 hours before staining cells with propidium iodide, running on a BD FACSCalibur and analyzing results using ModFit LT (N=3).