Sc 20682

For immunoblotting, extraction of proteins from cultured cells with a modified buffer was followed by immunoblotting with corresponding antibodies. Briefly, protein samples were fractionated on 10 - 15 % SDS polyacrylamide gels and electroblotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) using a semidry transfer apparatus (Bio-Rad). Rabbit polyclonal antibodies recognizing PARK2 (ab15954), were obtained from Abcam. Mouse monoclonal antibody recognizing PARK2 (sc-32282) and Lamin B1 (sc-20682) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibody recognizing NF-κB (sc-372) was obtained from Santa Cruz Biotechnology. Anti-β-tubulin and HA mouse antibodies were purchased from Sigma.

Total protein was extracted using Sodium dodecyl sulfate (SDS) reducing buffer (Biorad, CA, USA) and separated on 8–12% gradient SDS polyacrylamide gels and transferred on Polyvinylidene difluoride (PVDF) membranes (Biorad, CA, USA). Membranes were blocked with 5% non-fat dry milk for 30 min at room temperature, followed by overnight incubation with a primary antibody against caspase-1 (1:300, Santa Cruze) at 4 °C. Membranes were washed with PBS with 0.1% Tween 20 and incubated for one hour at room temperature with a secondary antibody (goat polyclonal anti-mouse IgG, ab205719, 1:3000) for one hour. Membranes were stained using anti-beta-actin (1:4000, A01546, GenScript) antibody to normalize protein expression for total protein and cytoplasmic fraction. Membranes were stained with anti-lamin b1 (1:300, sc-20682, Santa Cruz) antibody and secondary human, bovine, horse, horseradish peroxidase-conjugated goat anti-mouse IgGs (H&L) (A106PS; American Qualex) to normalize protein expression for nuclear proteins. Western blot results were visualized using Clarity Western ECL reagents (Biorad, CA, USA) and a ChemiDoc XRS + (Biorad, CA, USA).