Ab47563

Immunostaining was carried out as described by Montie et al., 2009 following 20 min fixation in 4% paraformaldehyde. Spinal cord tissue was embedded in OCT and cryosectioned (7μm) prior to fixation. All animal procedures were performed following the guidelines of the Office of Laboratory Animal Welfare and with the approval of the Thomas Jefferson University Institutional Animal Care and Use Committee.
Antibodies used include: AR (H280, Santa Cruz Biotechnology), AR (AR-318-CE, Leica Biosystems), AR pS650 (ab47563, Abcam), RanGAP1 (H180, Santa Cruz Biotechnology), Ran (610340, BD Transduction Laboratories), Lamin B1 (ab16048, Abcam), HDAC6 (D21B10, Cell Signaling Technology), HSP70 (SMC-100, StressMarq), neurofilament-heavy chain (SMI32; 801701 Sternberger Monoclonals), and 3B5H10 (a kind gift from Dr. Steve Finkbeiner, Gladstone Institute of Neurological Disease and UCSF). Immunostained cells were visualized using a Leica DMR Fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) and imaged using iVision Mac® or ProgRes® software.
In PC12 cells, the percentage of inclusions was determined by counting at least 300 cells in triplicate wells. Experiments were repeated at least three times.

Protein extracts were prepared by lysing cells in Triton-DOC lysis buffer (1% sodium deoxycholate, 0.5% Triton X-100 in PBS with protease inhibitors). All lysates were sonicated at least three times for 10 s using a Branson cup sonifier. Protein concentration was determined by DC protein assay (Bio-Rad). Lysates were electrophoresed by SDS-PAGE and transferred to 0.45 μm PVDF membrane (Immobilon-P). Blots were incubated with primary antibodies diluted in 5% nonfat milk in TBST (TRIS-buffered saline, 0.1% Tween-20) for 60 min at room temperature or overnight at 4 °C and with HRP-conjugated secondary antibodies (mouse: AP308P, rabbit: AP307B; Millipore) for 60 min at room temperature. Detection was performed using ECL substrate (Thermo Fisher) with analysis on a ChemiDoc MP (Bio-Rad).
Antibodies used include: AR (H280, Santa Cruz Biotechnology), AR pS650 (ab47563, Abcam), α-tubulin (21445, Cell Signaling Technology), β-catenin (8480 T, Cell Signaling Technology), Lamin B1 (ab16048, Abcam), p62 (ab56416, Abcam), and GAPDH (10R-G109A, Fitzgerald).