Ripa buffer

PC12 cells were pretreated with the control or GSs (20 μg/mL), followed by glutamate (15 mM/mL), for 24 h. Whole PC12 cell lysates were prepared in a radioimmunoprecipitation (RIPA) buffer (GenDEPOT, Katy, TX, USA) containing a protease and phosphatase inhibitor cocktail (GenDEPOT). Cell lysates containing equal amounts of protein were prepared using the Bradford assay. Proteins were mixed with 5× loading buffer and boiled for 10 min.

Protein were extracted from cells using RIPA buffer (GenDEPOT, TX, USA) containing 1% protease inhibitor and 1% phosphatase inhibitor cocktail (GenDEPOT) as lysis buffer. In total, 20 μg of extracted proteins is separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). Immunoblotting was performed as previously described by Gyamfi et al.^{4 (link)} List of all primary antibodies and dilutions used are indicated in Supplementary Table 6. All gels and blots were derived from the same experiment and were processed in parallel.

SMG-C6 cells were lysed in ice-cold RIPA buffer (GenDEPOT, Barker, TX; R4200-010) and protein concentrations were measured using s spectrophotometer (Nanodrop; Thermo Fischer Scientific, ND-1000). Protein samples were separated using 10% SDS-PAGE gels (Bio-Rad, Hercules, CA). After electrophoresis in a Power-Pac Basic system (Bio-Rad), proteins were transferred to nitrocellulose membranes using an iBLOT 2 Dry Blotting system (Thermo Fisher Scientific, IB21001). The membranes were blocked with 10% non-fat milk and incubated with anti-ERK antibodies (1:1000; Cell Signaling Technology, 9102) and anti-pERK antibodies (1:1000; Cell signaling, 9101) at 4 °C overnight. After washing, membranes were incubated with anti-rabbit IgG-HRP (1:5000; Santa Cruz Biotechnology, sc-2030). Immunoreactivity was visualized by ECL reagents (Thermo Fisher Scientific, 32106) and detected by the Chemidoc XRS+ system (Bio-Rad Laboratories).

Ins-1 cells (4 × 10⁵ cells/well) were seeded onto six-well plates, and the cells were incubated with DMSO (control) or 50 µM BPCA for 1 h and further incubated with or without 0.2 mM PA for 24 h. The cells were lysed using RIPA buffer (GenDEPOT, Barker, TX, USA) with a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) for 20 min on ice. The lysates were centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatant was used for Western blotting. The protein concentrations were measured using a DC^TM protein assay kit (Bio-Rad, Hercules, CA, USA). The following step was performed as described in our previous study [35 (link)]. The antibodies used for this experiment were purchased from Cell Signaling Technology (CST, Danvers, MA, USA) for anti-Bcl-2, anti-Bax, anti-cleaved caspase-3, anti-cytochrome C, and anti-parkin; anti-β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Proteins were extracted from PC-3 cells treated with different concentrations (0, 10, 30, 60, 90, and 270 μg/mL) of PWS and PIR extracts using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) (GenDEPOT, Baker, TX, USA). The cell lysate was separated on 8%–15% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA) treated with primary antibodies against cleaved caspase-3 (9661; Cell Signaling Technology, Danvers, MA, USA) and actin (sc-47778; Santa Cruz Biotechnology, Dallas TX, USA). After incubation with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, West Gove, PA, USA), signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Western blots were visualized using WSE-6100 LuminoGraph (ATTO, Tokyo, Japan).

Western blotting was performed as described [52 (link)]. The cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (GenDepot, Houston, TX, USA). The proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. The membranes were blocked in 5% non-fat milk in Tris-buffered saline-Tween 20 (TBS-T) and then incubated with the specific primary antibodies. After washed in TBS-T, the membranes were incubated with an HRP-conjugated secondary antibody. The bands were visualized by chemiluminescence. The following antibodies were used: antibodies against OTUD6A (1:1000, Proteintech, Rosemont, IL, USA), MYC (1:10,000, Proteintech, Rosemont, IL, USA), HSP90 (1:5000, Santa Cruz, Dallas, TX, USA), HA (1:1000, Santa Cruz, Dallas, TX, USA), FLAG (1:2000, Proteintech, Rosemont, IL, USA), Cyclophilin B (1:5000, Thermofisher, Waltham, MA, USA), Aurora-A (1:2000, Cell Signaling, Danvers, MA, USA), phospho-Aurora-A (Thr 288) (1:500, Abclonal, Woburn, MA, USA), PLK1 (1:1000, Abclonal, Woburn, MA, USA), and phospho-PLK1 (Thr 210) (1:1000, Cell Signaling, Danvers, MA, USA).