Nitrocellulose membrane

Cells were digested with low-concentration trypsin and collected into tubes. We used a Nuclear and Cytoplasm Protein Extraction Kit (Beyotime) to separate nuclear and cytoplasm proteins. Cells were lysed in an ice cold radio immunoprecipitation assay (RIPA) lysis buffer with 1 mM phenylmethyl sulfonyl fluoride (Sigma-Aldrich), which was used for cell protein extraction. Protein concentration was determined using a BCA KIT (Beyotime). Proteins were separated by 4%–12% SurePAGE gels (GenScript, Nanjing, China), transferred to a nitrocellulose membrane (Pall, Mexico), and then detected using antibodies according to standard procedures. Primary antibodies were applied overnight at 4 °C for western blot tests, and their dilutions were as follows: NCAPG (sc-101014, Santa Cruz, 1:1000); MYHC (MF20, Developmental Studies Hybridoma Bank, 1:50); MYOD (sc-377460, Santa Cruz, 1:1000); MYOG (sc-12732, Santa Cruz, 1:1000) and β-tubulin (10094-1-AP, Proteintech, 1:2000). Finally, secondary antibodies were visualized with HRP-conjugated secondary antibodies that were applied for 1 h at room temperature. ECL western blotting detection reagent (Beyotime) was used to visualize the protein bands.

To detect Hfq or RelA, protein samples were separated by either 15% acrylamide gel (RelA) or 4–20% MOPS gradient gel (Hfq). Thereafter, the proteins were transferred to nitrocellulose membrane (Genscript) by the Genscript eBLOT L1 fast wet protein transfer system, as suggested by the manufacturer. After transfer, the membrane was incubated at room temperature for 1 h (shaking) in blocking solution containing 4% BSA, 4% skim milk, and 1X TBST. The membrane was rinsed once with 1X TBST followed by incubation at room temperature for 1 h (shaking) in 10 ml of 1X TBST containing 3% BSA and 20 µl (1:500 dilution) of rabbit anti-Hfq raised against a synthetic peptide (SSAQNTSAQQDSEETE) of Hfq CTD (HY-LABS) or rabbit anti-RelA antibody raised against the purified RelA protein ADAR BIOTECH. Following three times wash (10 min each) with 1X TBST, the membrane was incubated in HRP conjugated secondary antibody solution (1 µl in 10 ml of 1X TBST, 1:10,000 dilution, Abcam, ab6721) for 1 h with shaking. The membrane was then rinsed once with 1X TBST and the protein were detected by incubation in ECL solution (Advansta Western Bright) for 1 min followed by the use of Image Quant LAS 4000 mini software.

Western blot was done as previously described (Coller and Parker 2005 (link); Sweet et al. 2012 (link); Zeng and Jin 2016 (link)). Briefly, fractions from polysome profiling were resolved by SDS–PAGE; and proteins in unfixed gels were transferred to nitrocellulose membranes (GenScript) for 30 min at 100 V in a Mini Trans-Blot apparatus (Bio-Rad). Protein bands were detected with anti-GST (Invitrogen) or anti-His antibodies (Sigma-Aldrich). Northern blots were done as previously described (Coller and Parker 2005 (link); Sweet et al. 2012 (link)). RNA samples were separated on a 5% urea acrylamide gel, transferred to nylon membranes, and probed overnight with a luciferase gene-specific ³²P-labeled DNA probe. Blots were exposed to PhosphorImager screens, scanned by a Storm 840 scanner, and quantified with ImageQuant software.