Anti smt3 y 84

SDS-PAGE was performed using 4–12% Bis-Tris gels (Novex). After running the gels, proteins were transferred onto nitrocellulose membrane (VitaScientific, DBOC80014). Western blotting was performed according to a standard protocol. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, 34078), or SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, 34580), or One Component Chemiluminescent Substrate (Rockland, UniGlow-0100) was used for the detection. Protein levels were quantified using Gene Tools software (version 3.8.8.0) from SynGene (Frederick, MD) or Image Lab software (version 6.0.0) from Bio-Rad Laboratories, Inc (Hercules, CA). Antibodies were as follows: anti-HA (12CA5) mouse (Roche, 11583816001), anti-HA rabbit (Sigma, H6908), anti-c-Myc (A-14) (Santa Cruz Biotechnology, sc-789), anti-Smt3 (y-84) (Santa Cruz Biotechnology, sc-28649), and anti-Tub2 antibodies (Basrai laboratory). Anti-HA was used at a dilution from 1:1000 to 1:5000, and anti-c-Myc, anti-Smt3, and anti-Tub2 were used at a dilution from 1:3000 to 1:5000. Secondary antibodies were ECL Mouse IgG, HRP-linked whole Ab (GE Healthcare Life Sciences, NA931V) or ECL Rabbit IgG, HRP-linked whole Ab (GE Healthcare Life Sciences, NA934V) at 1:5000 dilution.