Sc 133132

Cells were briefly washed twice, harvested in ice-cold PBS, and then collected by centrifugation at 2,500 × g at 4°C for 5 min. Cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 10% NP-40, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride) and incubated for 10 min on ice. After adding SDS loading dye, cell lysates were resuspended by a 26 gauge needle with a syringe. Protein samples were resolved by SDS-PAGE and transferred to the NC membrane (10600002; GE Healthcare, USA). The primary antibodies used in immunoblotting were rabbit anti-MRPL2 (HPA064814, 1:1,000; Atlas Antibodies, Sweden), mouse anti-HSP70 (sc-66048, 1:500; Santa Cruz Biotechnology), mouse anti-β tubulin (66240-1-Ig, 1:5,000; Proteintech, China), mouse anti-eIF2a (sc-133132, 1:500; Santa Cruz Biotechnology), and rabbit anti-phospho eIF2a (ab32157, 1:10,000; Abcam). Horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) were used for detecting primary antibodies and reacted with ECL solution (1705061; Bio-Rad, USA). Luminescence signals were detected by iBright FL1500 (Invitrogen).