Mabe343

The following antibodies were used for Western blot analysis and immunofluorescence microscopy: monoclonal mouse anti-p53 (DO-1, Santa Cruz, sc-126), polyclonal rabbit anti-p21-CIP1 (Santa Cruz, sc-397), monoclonal mouse anti-phospho-Ser139-H2AX (γH2AX) (JBW301, Millipore, 05–636), monoclonal mouse anti-MDM2 (Abcam, ab16895), polyclonal rabbit anti-phospho-Ser51-eIF2α (Cell Signaling, #9721), polyclonal rabbit anti-eIF2α (Cell Signaling, #9722), monoclonal mouse anti-puromycin (Millipore, MABE343), polyclonal rabbit anti-β-actin (Abcam, ab8227-50), polyclonal rabbit anti-TOB1 (Proteintech, 14915-1-AP), monoclonal rabbit anti-RPS20 (Abcam, ab133776) and monoclonal rabbit anti-Cyclin D1 (EPR2241, Abcam, ab134175).

Synchronous arrested L1 larvae were obtained as described above. A total of 16000 L1 larvae were grown until the young adult stage on NGM plates seeded with E. coli OP50 bacteria. Animals were washed, grown and treated with puromycin as previously described [29 (link)]. For the Western blot analysis, three micrograms of total protein were used per well. Incorporation of puromycin into nascent peptides was measured by normalizing band intensity from monoclonal anti-puromycin (Merck, MABE343) antibody to anti-actin (Abcam, ab8227).

Pellets containing a mix of embryos, larvae and adults were prepared as described above. Lysates were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in lysis buffer (50 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl₂, 0.1% Triton X-100, 5% glycerol (w/vol), 1 mM PMSF, 7 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001). Lysates were cleared by centrifugation at 16100 x g for 20 minutes at 4°C. Protein concentrations were approximated by Bradford Assay (Bio-Rad). Samples were prepared by mixing 25 micrograms of protein extract with the required amount of 4x NuPAGE™ LDS Sample Buffer (Invitrogen, NP0007) and 10x NuPAGE Sample Reducing Agent (Invitrogen, NP0004), followed by heating at 70°C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane by wet-transfer. The following primary antibodies were used: 1:1000 monoclonal mouse anti-eEF1A (Merck, 05–235), 1:10000 monoclonal mouse anti-puromycin (Merck, MABE343), 1:5000 polyclonal rabbit anti-actin (Abcam, ab8227). Detection was carried out with IRDye 680RD-conjugated goat anti-mouse secondary antibody (LI-COR Biosciences, 926–68070) or IRDye 800CW-conjugated goat anti-rabbit secondary antibody (LI-COR Biosciences, 926–32211) and infrared imaging (LI-COR Biosciences, Odyssey CLx).