FIG. 9.

Decreased eIF4E expression during myeloid differentiation correlates with decreased levels of the 4E regulatory factors, c-Myc and protein synthesis. (A and B) For Southwestern analyses, protein lysates from U937 (A) and HL60 (B) cells were prepared with Laemmli buffer at 0, 3, 6, 24, 48, and 72 h after the addition of TPA. The lysates (50 μg) at the indicated time points were analyzed with the radioactively labeled LS3 trimer oligonucleotide in a Southwestern assay (SW panels). Size markers (lane SM) are indicated. For Northern blots, total cellular RNA was harvested from U937 (second through fourth panels in panel A) and HL60 (second through fourth panels in panel B) cells after the addition of TPA. RNA was size fractionated, run on formaldehyde-agarose gels, blotted, and hybridized with eIF4E, c-myc, or GAPDH plasmid fragments (4E, myc, and GAPDH, respectively). The protein lysates used for the Southwestern analyses were additionally run on 10% denaturing polyacrylamide gels, blotted, and probed with anti-eIF4E, anti-c-Myc, and anti-actin antibodies for the U937 (fifth through seventh panels [4E, myc, and actin] in panel A) and HL60 (fifth through seventh panels [4E, myc, and actin] in panel B) cells. (C through F) U937 (C and E) and HL60 (D and F) cells were pulse-labeled for 3 h with [³⁵S]methionine and [³H]thymidine at the indicated time points. Aliquots of protein lysates at each time point were harvested directly in Laemmli buffer and run on 10% denaturing polyacrylamide gels to simultaneously evaluate protein synthesis rates of multiple individual proteins (C and D). Counts incorporated during pulse labeling were further evaluated by trichloroacetic acid precipitation of cell lysates (E and F). [³⁵S]methionine (solid bars; y axis on right) and [³H]thymidine (open bars; y axis on left) incorporation is displayed as the mean and standard deviation of four determinations at each time point to evaluate the regulation of net protein synthesis (³⁵S) and DNA synthesis (³H) during differentiation.