Criterion xt precast gel

Yolk protein levels were quantified by running worm protein extracts on PAGE gels and then staining with Coomassie blue dye as described [19 (link)]. 20 worms were picked into 25 μL of M9 buffer and frozen at −80°C. Samples where then thawed, and 25 μL of 2x Laemmli sample buffer (Sigma) added. Samples were incubated at 70°C, vortexed continuously for 15 min, incubated at 95°C for 5 min and spun at 6,000 rpm for 15 min. Samples were loaded onto Criterion XT precast gels 4%–12% Bis-Tris (Bio-Rad), using XT MOPS (Bio-Rad) as a running buffer, and stained and destained following standard protocols. Gels were analyzed using ImageQuant LAS-4000 (GE Healthcare). Protein band identification was based on published data [19 (link), 21 (link)]. YP170, YP115 and YP88 were previously identified as vitellogenins by means of peptide mapping [60 (link)]. Correct identification in the present study of vitellogenins on protein gels was confirmed by the effect of vit-5,6 RNAi, which abrogated YP170, YP115 and YP88 accumulation (Figure S4A). Yolk proteins were normalized to myosin and the ratio of actin to myosin was estimated to assess the reliability of myosin as a standard for normalization [19 (link)].

Cell lysates were collected in Laemmli buffer [0.05 M Tris-HCl (pH 6.8), 1% SDS, 10% glycerol and 0.1% β-mercaptoethanol] and denatured for 10 min at 95°C, quantified using BCA quantification (Thermo Fisher Scientific), resolved using CriterionXT pre-cast gels (BioRad) and transferred to nitrocellulose membranes. Blocking was performed in 5% milk in TBST (TBS-Tween) buffer for 1 h at room temperature. Primary antibody incubation (p53, p21, Bcl2, Tubulin - see Table S4) was carried out overnight at 4°C in TBST containing 5% BSA. Membranes were washed three or four times in TBST at room temperature with gentle agitation and then incubated with the secondary antibody (HRP-conjugated, anti-mouse or anti-rabbit) in blocking solution (5% milk in TBS-T) at 1:5000 dilution. Membranes were then washed three or four times in TBST at room temperature with gentle agitation. Western blot quantification was performed using Fiji software version 2.0.0-rc-49/1.51d. Protein expression levels were normalized to loading control tubulin.

Western blot analysis was performed as described [36 (link)]. Equal amounts of protein (15–20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane using Criterion XT Precast gels (Bio-Rad, USA). Membranes were blocked with 5% BSA for 2 h; then the corresponding antibodies GLUT4 (1 : 1000, #347063, Zen Bio), HK2 (1 : 1000, #200569, Zen Bio), PFKM (1 : 1000, #ab154804, Abcam), PKM (1 : 1000, #200667-1A7, Zen Bio), LDHA (1 : 500, #384822, Zen Bio), AMPKα (1 : 1000, #ab207442, Abcam), p-AMPKα (1 : 1000, #AP0116, ABclonal), mTOR (1 : 1000, #380411, Zen Bio), p-mTOR (1 : 1000, #5536, Cell signalling), 4EBP1 (1 : 500, #306002, Zen Bio), p-4EBP1 (1 : 500, #ab278686, Abcam), and Actin (1 : 2000, #200068-8F10, Zen Bio) were incubated overnight at 4°C; finally, blots were incubated with secondary antibodies at room temperature for 2 h. Bands were visualized with the gel imaging system (Syngene, UK) and analyzed with ImageJ analysis software (National Institutes of Health, https://rsb.info.nih.gov/ij/). The research met all the requirements of ARRIVE checklist and most of the requirements of CONSORT checklist if applicable (see supplemental files, named S1 ARRIVE Checklist and S2 CONSORT 2010 Checklist).