Rpl24

The proteins from LSCC tissues were isolated as previously described (Method 2.2). For the cell lines, we harvested and thrice washed cells with PBS. They were then lysed on ice for 30 min in RIPA buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease and phosphatase inhibitors. Afterwards, protein separation was conducted via SDS-PAGE, and their transfer onto a PVDF membrane followed. The mem-brane underwent blocking with 5% nonfat milk before overnight incubation with primary antibodies at 4 °C. The employed primary antibodies included GAPDH (Abclonal, Wuhan, China, AC001), β-actin (Abclonal, A17910), RPS10 (Abclonal, A6056), CDH1 (Abclonal, A24874), CDH2 (Abclonal, A19083), Snail (Abclonal, A5243), RPL29 (Immunoway, Plano, TX, USA, YN0379), RPS28 (Immunoway, YN4647), RPL3 (Immunoway, YT4105), RPS26 (Proteintech, Wuhan, China, 14909), VIM (Proteintech, 10366), FN (Proteintech, 15613), ZO1(Proteintech, 21773), and RPL24 (Proteintech, 17082). After being rinsed with PBS and 0.1% Tween 20, the membrane was incubated with the pertinent secondary antibodies for an hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents and the Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA), with their quantification performed using ImageJ software (version 1.49).

Cells were lysed in RIPA buffer (10 mM Tris-HCL (pH 8.0), 1mM EDTA, 0.5 mM EGTA, 1% triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 20 mM NaF, Complete EDTA-free protease inhibitor tablets (Roche Diagnostics Corp., Basel, Switzerland) and the phosphatase inhibitor cocktail PhosSTOP (Roche)), the latter two as indicated by the manufacturer's protocol. Equal amounts of protein were diluted in 2X sample buffer. Immunoblots on PVDF (Polyvinyldene Fluoride) membranes were blocked with nonfat milk in tris-buffered saline with 0.05% tween-20 (TBST). The following antibodies were incubated with membranes in 5% nonfat milk in TBST: RPL24 (Proteintech, Chicago, IL), Cyclin D1, Rack1, RPL4 (Santa Cruz Biotechnology, Santa Cruz, CA), NBS1, GAPDH (EMD Millipore Corporation, Chicago, IL), Survivin, β-tubulin, acetyl-lysine, eIF6, acetyl-H2B, H2B, (Cell Signaling Technology, Boston, MA), acetyl-tubulin, tubulin (Sigma Aldrich (St. Louis, MO)). Densitometry was performed using ImageJ software.