Western Blot Protein Quantification Protocol

Protein extracts from either total homogenates or subcellular fractions were used for Western blot assays with standard procedures. Nuclear and cytoplasmic fractions were separated using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Fifteen micrograms of protein concentrate were separated on 12% SDS–PAGE gels at 120 V and transferred onto PVDF membranes (Pall Life Sciences) at 250 mA for 90 min. After transfer, the PVDF membranes were blocked with 3% BSA/0.1% Tween-TBS buffer for 1.5 h and incubated overnight at 4°C with the following primary antibodies: mouse ELAVL1/HuR (dilution 1:1,000; Santa Cruz) and mouse HSP70 (dilution 1:2,000; Santa Cruz). As a secondary antibody, we used an HRP-conjugated goat anti-mouse antibody (dilution 1:10,000, Abcam). An HRP-conjugated alpha tubulin antibody (dilution 1:2,000, Rockland) was used as a loading control. The membrane signals were detected using chemiluminescence (ChemiDoc MP, Bio-Rad). Protein bands were quantified using ImageJ software with the Band/Peak Quantification Tool (see text footnote 1).