Proteoprep membrane extraction kit

Total protein, membrane protein, cytoplasmic protein, and nucleoprotein were extracted from RA FLS and HEK293T cells by ProteoPrep® Total Extraction Sample Kit (Sigma-Aldrich), ProteoPrep® membrane Extraction Kit (Sigma-Aldrich), and NE-PER™Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), respectively. Protein in culture medium (100 ml) was pretreated by freeze-drying method and then dissolved in an appropriate amount of double distilled water. After quantification with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), 25 μg protein samples were separated by SDS-PAGE and then electrotransfered onto polyvinylidene difluoride membranes. After blocked in 5% skim milk for 2 h, the membranes were incubated with antibodies of KIAA1199 (#ab98947, Abcam), HYAL2 (#ab126099, Abcam), CD44 (#ab157107, Abcam), AXNA1 (#ab88865, Abcam), PI3K (#4249, CST), total-Akt (#2920, CST), p-Akt (Ser473) (#4060, CST), total-STAT3 (#4904, CST), p-STAT3 (Tyr705) (#9131, CST), p-P65 (Ser536) (#3036, CST), and then with matching HRP-conjugated secondary antibodies. Protein bands were visualized using ECL substrate (#35055, Thermo Fisher Scientific). β-Actin (#AP0060, Bioworld) and Lamin B1 (#12987-1-AP, Proteintech) were used as the internal reference of cytoplasmic protein and nucleoprotein, respectively. The intensity of protein bands were analyzed by Image J (v1.8.0).

For immunoprecipitation, 10⁶ hFLSCs per sample were incubated with 200 ng/mL His-tagged TNF-α, FasL, or TRAIL (R and D Systems) for 30 min at 37°C, followed by cross-linking with 5 mM BS³ and 20 mM quench solution (pH 7.5). Then, the cells were washed twice with cold PBS and lysed using ProteoPrep membrane extraction kit (Sigma-Aldrich) in the presence of protease and phosphatase inhibitor cocktails. To prepare denatured proteins for immunoprecipitation, cells were lysed with buffer containing 50 mM of Tris (pH 7.9), 10 mM of dithiothreitol, 5 mM of EDTA, and 1% of SDS and then boiled for 10 min. The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C. Samples were treated with 40 μL protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membranes (Millipore, Billerica, MA) for western blotting.

We collected SW1990 cells using a ProteoPrep Membrane Extraction Kit (Sigma), after which the cells were suspended in a low conductivity buffer and disrupted by ultrasonication and other appropriate methods. The membranes and membrane proteins were centrifuged, washed with water and resuspended in a chaotropic buffer solution. This protein solution was then reduced and alkylated. The end result was a soluble membrane protein sample that contained low salt levels and was ready for separation using isoelectric focusing (IEF). After extraction, the membrane proteins were immediately frozen at −80 °C.