Goat anti mouse igg hrp antibody

The transfected cells were lysed in 100 μL of a RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, cocktail protease inhibitor (Roche Applied Science)) on ice for 30 min and centrifuged at 15,000 g for 15 min. The supernatant was added with 100 μL of the loading buffer (2% SDS), while the pellet was sufficiently washed with the RIPA buffer for three times and then added with 50 μL of the loading buffer (4% SDS). Equal volume of supernatant and pellet fractions was subjected to SDS-PAGE with 12% acrylamide gel and transferred onto PVDF membranes (PerkinElmer). The mouse monoclonal antibodies against FLAG or Myc, goat anti-actin (Santa Cruz), goat anti-mouse IgG-HRP antibody and rabbit anti-goat IgG-HRP antibody (Jackson Immuno-Research) were used. The proteins were detected by an ECL detection kit (Thermo scientific). The band intensities were quantified by Scion Image software (Scion Corp).

17-AAG (17-(Allylamino)-17- demethoxygeldanamycin) and MG132 were purchased from Sigma and Calbiochem, respectively. The antibodies against HA, FLAG and endogenous CHIP were obtained from Sigma, while those against HSP90 and Myc were from Cell Signaling and those against GFP, ubiquitin and actin from Santa Cruz. The anti-USP19 antibody (A301-587A) was purchased from Bethyl Laboratories. The goat anti-mouse IgG-HRP antibody, goat anti-rabbit IgG-HRP, rabbit anti-goat IgG-HRP secondary antibodies and FITC-conjugated anti-mouse antibody, and Cyanine 3 conjugated anti-rabbit secondary antibody were purchased from Jackson Immuno-Research. The proteins were visualized using an ECL detection kit (Amersham Pharmacia Biotech). CytoTox-ONE^™ reagent was a product of Promega. Human USP19_a, USP19_b, USP5 and all the mutants were cloned into HA-pcDNA3 vector. The mutants (C506S, CS1M, CS2M, CS12M, ΔN393) of USP19_b, C335A mutant of USP5 and the Atx3_100Q-UIM^mut (S236A/S256A) mutant were generated using site-directed mutagenesis via PCR technique. The expression plasmid for human HSP90 was cloned into pcDNA3.1-Myc/His, and the pEGFP-N1 vector was used to express EGFP. The polyQ proteins (including Atx3_22Q, Atx3_100Q, Atx3_100Q-UIM^mut, Htt-N552_18Q, Htt-N552_100Q and Htt-N171_100Q) and TDP-35 were cloned into pcDNA3-FLAG plasmid.