Figure 1.
Analysis of YB-1 and YB-1 mRNA amounts in the cell.
A and B, 15 µg of total protein from lysates of various cell lines (A) or 50 µg of total protein from tissue lysates (B) were analyzed by Western-blotting. C and D, 10 µg of total RNA from lysates of various cell lines (C) or tissue lysates (D) were analyzed by Northern-blotting.
Figure 2.
Analysis of YB-1 mRNA distribution between polysomal and free mRNP fractions in various cell lines. A,
Cells were scraped and lysed. Nuclei and mitochondria were removed by centrifugation, and cytosolic extracts were then spun through a 50% sucrose cushion at 100,000 rpm in a TLA-100 centrifuge (Beckman) for 13 min to separate postpolysomal supernatant from polysomes. Total RNA from postpolysomal supernatant and polysomal fractions (resuspended pellets) were extracted with TRIzol, subjected to agarose gel electrophoresis and Northern blot hybridization to [32P]-labeled YB-1 and GAPDH cDNA. B, Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The sum of relative radioactivity values in free and polysomal mRNP fractions was taken to be 100%.
Figure 3.
Analysis of YB-1 mRNA distribution between polysomal and free mRNP fractions in rabbit organs. A,
Tissue cytosolic extracts were spun through a 50% sucrose cushion at 100,000 rpm in a TLA-100 centrifuge (Beckman) for 13 min to separate postpolysomal supernatant from polysomes. Total RNA from postpolysomal supernatant and polysomal fractions (resuspended pellets) were extracted with TRIzol, subjected to agarose gel electrophoresis and Northern blot hybridization to [32P]-labeled YB-1 and GAPDH cDNA. B, Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The sum of relative radioactivity values in free and polysomal mRNP fractions was taken to be 100%.
Figure 4.
Assessment of YB-1 synthesis in the cell. A
. HeLa cells were labeled with [35S]-methionine for 2 h, harvested and lysed. Cell lysate was used for immunoprecipitation with preimmune antibody or YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, stained with CBB G250, and the [35S]-labeled proteins were detected by autoradiography. Protein with an electrophoretic mobility corresponding to the recombinant YB-1 was cut out from the gel and identified by mass-spectrometry as YB-1. B, Assessment of YB-1 synthesis in cells of various lines. Cells were labeled using [35S]-methionine, harvested and lysed. Cell lysates were counterbalanced by radioactivity and used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and the [35S]-labeled proteins were detected by autoradiography.
Figure 5.
Dependence of YB-1 synthesis on cell confluence.
NIH3T3 and HEK293 cells of various confluence were [35S]-methionine-labeled for 1 h, harvested and lysed. Cell lysates were counterbalanced by radioactivity (A and C) and used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [35S]-labeled proteins were detected by autoradiography (B and D). Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.).
Figure 6.
Renewal of YB-1 amount and YB-1 synthesis after serum starvation release.
3T3 (A) and HEK293(B) cells were serum starved (2 days). Cells were harvested at indicated time intervals after serum addition, lysed and used for Western blot analysis. 3T3(C) and HEK293(D) cells were serum starved (2 days). Control cells, serum starved cells and serum stimulated (6 h) cells were labeled with [35S]-methionine for 2 h, harvested and lysed. Cell lysates were used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [35S]-labeled proteins were detected by autoradiography. Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The level of YB-1 synthesis in cells without serum starvation was taken to be 100%.
Figure 7.
Effect of mTOR inhibitor PP242 on endogenous YB-1 synthesis in the cell.
Untreated or PP242-treated (1 µM) HeLa (A, B, C, D) and HEK293 (E, F, G) cells were labeled with [35S]-methionine for 2 h, harvested and lysed. Cell lysates were counterbalanced by the total protein (B and E) and used for Western blotting (A) or immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and the [35S]-labeled proteins were detected by autoradiography (C and F). Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.) (D and G). The level of YB-1 synthesis in cells without PP242 treatment was taken to be 100%.
Figure 8.
Effect of various cell signaling pathway inhibitors on endogenous YB-1 synthesis in the cell.
Untreated (lane 1) or 1 µM PP242-treated (lane 2), or 0.1 µM rapamycin-treated (lane 3), or 0.5 µM wortmannin-treated (lane 4), or 10 µM U0126-treated (lane 5) HeLa cells were labeled with [35S]-methionine for 2 h, harvested and lysed. Cell lysates were counterbalanced by the total protein (A) and used for Western blotting (D) or immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [35S]-labeled proteins were detected by autoradiography (B). Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.) (C). The level of YB-1 synthesis in cells without drugs was taken to be 100%.
Figure 9.
Effect of PP242 and other cell signaling pathway inhibitors on reporter mRNA translation in the cell. A.
Scheme of mRNAs used here. rYB-1 mRNA 5′ UTR denotes rabbit YB-1 mRNA 5′ UTR (118 nt), hYB-1 mRNA 5′ UTR denotes human YB-1 mRNA 5′ UTR (171 nt), GAPDH mRNA 3′ UTR stands for Glyceraldehyde 3-phosphate dehydrogenase mRNA 3′ UTR. B. Untreated or PP242-treated (1 µM) HeLa cells were transfected by indicated reporter Firefly luciferase mRNA and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase (Fluc and Rluc, respectively). C. Untreated or PP242-treated (1 µM), or rapamycin-treated (0.1 µM), or wortmannin-treated (0.5 µM), or U0126-treated (10 µM) HeLa cells were transfected by reporter Firefly luciferase mRNA (rYB-1-luc-YB-1) and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase. The Fluc/Rluc ratio for the control (cells without treatment) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations.