Figure 1
OLA1 suppresses mammalian protein synthesis.
(A) Distribution of OLA1 in 10–50% sucrose density gradient fractions of A549 cell extract as measured by IB. Distributions of rpS6 (a small subunit protein), rpL26 (a large subunit protein) and eIF2α are shown for comparison. β-actin and Cyclin D1 were used as negative controls. Positions of the S40, S60, S80 and polysome fractions are also indicated. (B) The effect of OLA1 and control proteins (RFP and actin) on protein synthesis in RRL with luc mRNA as translation template (n = 3). (C) Effect of OLA1 overexpression and KD on mRNA translation in vivo. A diagram of the bicistronic reporter (rLuc-IRES-ffLuc which mediates the cap-dependent translation of Renilla luc and IRES-dependent translation of firefly luc) is shown (top panel). HeLa cells were transfected with OLA1-expression vectors (middle) or OLA1-siRNA (bottom) for 48 h, followed by transfection of the reporter for 24 h (n = 3). OLA1 expression was evaluated with IB (right). (D) Radiolabeling of de novo protein synthesis in OLA1-KD MDA-MB-231 cells. After 72 h starvation, serum was restored for indicated periods in the presence of [35S]Met/Cys (n = 3). Error bars: SD, Student’s t test: *p < 0.05; **p < 0.01; NS, not significant. Cropped blots are used. Full scan images of immunoblots are presented in Figure S10.
