Ne per nuclear and cytoplasmic protein extraction kit

To extract nuclear proteins, protein extracts were prepared from andrographolide-treated GBM8401 cells by using the NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology, Rockford, IL, USA).

Cells were washed with ice-cold PBS and lysed with RIPA buffer (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail and centrifuged at 13,000× g at 4 °C for 20 min. Nuclear proteins were isolated using the NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentration was determined with the BCA Protein Assay kit (Pierce Biotechnology). Next, 30 μg of total soluble proteins or 5 μg of nuclear proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Merck-Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies (1:1000 dilution) described in Materials, followed by incubation with appropriate secondary antibodies (1:5000 dilution). The immunoreactive bands were visualized with a West-Q chemiluminescent Substrate kit (GenDEPOT, Barker, TX, USA) and the band intensities on films were analyzed by densitometry to quantify protein expression using a FluorS MultiImager (Bio-Rad, Hercules, CA, USA). The membranes then were washed with Restore Western Blot Stripping Buffer (Thermo Scientitis, Waltham, MA, USA) and reprobed with 1:1000 diluted anti-GAPDH polyclonal or anti-Lamin B polyclonal antibodies to normalize for cytosolic or nuclear protein loading, respectively.

For nuclear and cytosolic protein extraction, protein extracts were prepared from HOS cells treated with 50 or 100 μM RESV using an NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology, Rockford, IL).

For Western blotting, cell lysates were prepared in RIPA buffer [1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Roche)], and then were subject to standard procedures of SDS-PAGE and antibody detection [26 (link)]. Antibodies used were: β-actin (AC-74, 1:5000, Sigma), MMP7 (monoclonal, JL07, sc80825, 1:500, Santa Cruz), p14^ARF (14P02, 1:750, NeoMarkers), E-Cadherin (sc33743, 1:500, Santa Cruz). Cell fractionation was performed using NE-PER nuclear and cytoplasmic protein extraction kit or subcellular protein fractionation kit (Thermo Scientific) followed by standard procedures of SDS-PAGE and antibody detection for IB using primary antibodies to: MMP7 (monoclonal, JL07, sc80825, 1:500, Santa Cruz or ab176325, 1:1000, Abcam), PARP (9542, 1:1000, Cell Signaling), HSP90 (H114, sc-7947, 1:1000, Santa Cruz).