Extended Data Fig. 5: eIF2B5 depletion regulates IRES-mediated translation of MYC.
(a) Immunoblot of SW480 cells after DMSO (-) or cymarine ( + ) treatment (100 nM, 24 h), representative of two independent experiments with similar results. Quantification of MYC, Cyclin E and c-Fos, relative to Vinculin, is shown below the immunoblot. (b) Schematic diagram of the MYC IRES sequence. Position of used sgRNAs for deletion of the IRES as well as primers for detection by PCR and expected size of the PCR products are indicated. (c) Agarose gel electrophoresis of PCR products from MYC IRES undeleted (CTR) and deleted (sgMYC IRES) cells. The primer pair used is shown in (b). PCR was performed once as control for deletion. (d) Immunoblot of control (CTR) and MYC IRES deleted (sgMYC IRES) APCdef and APCres cells, transduced with shCTR or shEIF2B5 #3 (72 h ethanol or doxycycline, respectively), representative of two independent experiments with similar results. (e) mRNA expression of ATF3 and GADD34 in shCTR and eIF2B5-depleted APCdef and APCres cells upon MYC depletion (96 h ethanol or doxycycline, respectively). siRNA transfections were carried out using siCTR as non-targeting control or siMYC for 72 h. Data represent mean ± s.d. (n = 3 technical replicates), representative of two independent experiments with similar results. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 5.
