Proteoextract subcellular proteome extraction kit

Neuro2a cells were transfected with the indicated plasmids. At 24 h after transfection, cells were collected by a scraper in 300 μl PBS buffer. Cell lysates were centrifuged at 500 g for 10 min at 4°C. After aspiration of the supernatant, cells were treated by one freeze–thaw cycle and assayed with ProteoExtract Subcellular Proteome Extraction kit (Millipore, 549790) according to the manufacturer's protocol. Cell lysates were fractionated into cytosol, organelles (including mitochondria), nuclei, and cytoskeleton (including insoluble matter).

Cells were collected from cultured dishes and lysed in RIPA lysis buffer (Beyotime, Hangzhou, Zhejiang, China) supplemented with protease inhibitors or phosphatase inhibitors. Cytoplasmic, mitochondrial, and nuclear protein fractions were extracted using ProteoExtract Subcellular Proteome Extraction Kit (Millipore, Billerica, MA). Protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime, Hangzhou, Zhejiang, China). Cell lysates (40 μg protein/line) were separated via 6%-15% SDS-PAGE and transferred to 0.45-μm thick polyvinylidene difluoride (PVDF) membranes (Millipore). The blotted membranes were blocked with 5% skim milk for 1 h at room temperature. Afterward, membranes were incubated with primary antibodies (1:1,000) overnight at 4 °C and then with HRP-labeled secondary antibody (1:2,000) for 1 h at room temperature. All the antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA). Detection was performed on a Fujifilm Las-4000 Luminescent Imaging System using ECL Kit (Pierce, Rockford, IL).

The cells were fractionated for membrane and cytosol fractions using ProteoExtract® Subcellular Proteome Extraction Kit per the manufacturer’s protocol (Millipore Sigma). After specific treatments, cells were washed and incubated cells with ice-cold Extraction I containing 5 μl of protease inhibitor mixture for 10 min at 4 °C with gentle agitation. To obtain the cell supernatant containing cytosol fraction, the above suspension was centrifuged at 1000 g at 4 °C for 10 min. The leftover pellet was resuspended in 1 mL of ice-cold Extraction II containing 5 μl of protease inhibitor mixture and incubated for 30 min at 4 °C. After centrifugation at 1000 g for 10 min at 4 °C, the supernatant (membrane/organelle fraction) was used to detect GRP78 protein expression.

Nuclear proteins were isolated using a ProteoExtract^® Subcellular Proteome Extraction Kit (539790, Millipore, State, Country). The nuclear protein fraction was separated using SDS-PAGE and analyzed using a conventional Western blot assay as described elsewhere (Zhou et al., 2014 (link)). Immunofluorescence staining was performed as follows. A total number of 50,000 cells were seeded in six wells and allowed to attach overnight. Adherent cells were fixed with 4% formaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for ten minutes, and blocked with 5% BSA for 30 min. They were incubated with primary antibodies at 4°C overnight. Subsequently, Alexa Fluor 568 labeled antibody was applied for 1 h at room temperature. Cell nuclei were counterstained with DAPI. Immunofluorescence images were acquired using a Leica DMRE microscope with HiPic software (Leica, Bensheim, Germany).

Liver tissue (50 mg) obtained from mice was washed twice with minimal essential medium. Subcellular fractions were prepared using the ProteoExtract subcellular proteome extraction kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Cytosolic, organelle/membrane, and nuclear proteins were collected into fractions 1 (F1), 2 (F2), and 3 (F3), respectively. The protein concentrations were quantified using the DC protein assay. Each fraction (3 μg protein) was separated using SDS-PAGE, followed by western blotting.

HCT-116^p53+/+ and HCT-116^p53−/− cells were treated either with DMSO as a negative control or with 15 μM XCT790 for 48 h. Afterwards, cells were digested with trypsin and harvested in PBS. Cytosolic, membrane/organelle, and nuclear fractions were isolated using ProteoExtract® subcellular proteome Extraction kit (Millipore, Danvers, MA, USA) following the manufacturer’s suggested protocols. To screen differential protein expression across the samples in membrane/organelle fractions, MS/MS^ALL with SWATH^TM acquisition was conducted. Raw data was collected through information-dependent acquisition (IDA) and SWATH^TM acquisition Sciex TripleTOF™ 5600 with Eksigent 1D+ nano LC. The protein identification, MS peak extraction, and statistical analysis were performed with ProteinPilot^TM (version 4.5), PeakView^TM (version 2.2), and MarkerView^TM (version 1.2), respectively.

The WB procedure was performed as described previously
[39 (link)] with some modifications. Briefly, after transient transfection with plasmids, cells were harvested and lysed in a lysis buffer containing a cocktail of protease inhibitors. After centrifugation at 12,000 rpm for 15 min at 4°C, supernatants were collected, mixed with dithiothreitol, and used for WB. The ProteoExtract® Subcellular Proteome Extraction Kit (Millipore) was used for extraction of subcellular fractions following the manufacturer’s protocol. Equal amounts of protein extract were electrophoresed in 10% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, incubated with the primary antibody overnight at 4°C, and incubated with the secondary antibody at room temperature for 1 h. Blots were washed in Tris-buffered saline with 0.1% Tween-20 and proteins were visualized by chemiluminescence. The following antibodies were used for WB: anti-SOX1 (IB 1:2000, Epitomics, CA, USA), anti-CK19 (1:8000, Epitomics), anti-CK18 (1:4000, Epitomics), anti-CK13 (1:4000, Epitomics), anti-CK8 (1:4000, Epitomics), anti-Involucrin (1:4000, Abcam, MA, USA), anti-GAPDH (1:10000, ProteinTec Group, IL, USA), anti-c-Myc (N-262) (1:2000, Santa Cruz Biotechnology, CA, USA), and anti-β-catenin (1:2000, Upstate, NY, USA).