Horseradish peroxidase hrp conjugated secondary anti rabbit or anti mouse igg antibodies

Cultured cells were harvested and resuspended in Roeder D (200 mg/ml glycerol, 100 mM KCl, 0.2 mM EDTA, 100 mM Tris pH 8.0, 500 μM DTT and 200 μM PMSF). Cell lysis was carried out in a Bioruptor® Plus sonication device (Diagenode) for 10 min (low intensity settings, 30 s on/off). Sixty micrograms of proteins in cell lysates were separated on a NuPAGE™ 4–12% Bis-Tris Protein Gel (Invitrogen) and transferred onto a nitrocellulose membrane (GE) in a GENIE® blotter (Idea Scientific) at 12 V for 1 h. The membrane was blocked with 1:10 Western Blocking Reagent (Roche) in TBST (20 mM Tris pH 7.5, 137 mM NaCl and 0.1% (v/v) Tween 20). Proteins were detected with the following primary antibodies in TBST containing 1:20 Western Blocking Reagent, including rabbit polyclonal anti-HuR (Millipore), rabbit polyclonal DHX9 antibody (Proteintech), monoclonal anti-α-tubulin antibody (Sigma-Aldrich) and purified mouse anti-α-Synuclein (BD Biosciences). Following three washes in TBST, the membrane was incubated in the horseradish peroxidase (HRP) conjugated secondary anti-rabbit or anti-mouse IgG antibodies (Cell Signalling Technology) and developed with chemiluminescent substrate (Thermo #34580). Quantification of western blot bands were carried out in Image Studio Lite Ver 5.2.