Cheiscope 3300 mini

Northern blotting for select miRNAs was performed as previously described [75 (link)]. Briefly, approximately 10 μg of total RNAs were denatured at 70 °C for 10 min and then resolved in a 15% (W/V) TBE-urea polyacrylamide gel by electrophoresis at 150 V for approximately 1 h. The separated RNAs were transferred from the PAGE gel to a nylon membrane (Hybond-NX, GE Healthcare, USA) with the semi-dry method and subjected to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-mediated crosslinking at 65 °C. For probe preparation, DNA oligonucleotides complementary to targeted miRNAs were synthesized and labeled with biotin molecules at both 5′ and 3′ ends (Additional file 18: Table S17). After hybridization, the probes were detected with the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher, USA) following the manufacturer’s instructions. Signals were visualized by CheiScope 3300 Mini (CLINX, China). At least two biological repeats were performed for each miRNA.

Western blotting was performed to investigate ribosomal protein L13 (RPL13) and phosphoenolpyruvate carboxylase (PEPC) levels in polysomal fractions according to the method described by Li et al. [19 (link)]. Briefly, approximately 10 μg of total proteins from Total, TP, and MBP samples were subjected to separation in an 8% (W/V) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) by electrophoresis at 110 V for about 1 h. After proteins were transferred from the PAGE gel to a nitrocellulose membrane (Amersham Protran, GE Healthcare, USA), RPL13 and PEPC were detected with rabbit anti-RPL13 primary antibody (AS13 2650, Agrisera, USA) and rabbit anti-PEPC primary antibody (AS09 458, Agrisera, USA), respectively. The membranes were then incubated with goat anti-rabbit HRP-conjugated secondary antibody (AS09 602, Agrisera, USA). Blotting signals for RPL13 and PEPC were visualized by incubation with Agrisera ECL SuperBright detection reagent (AS16 ECL-S, Agrisera, USA) followed by exposure to CheiScope 3300 Mini (CLINX, China). Two biological repeats were performed for RPL13 and PEPC, respectively.