Ripa lysis buffer

LNCaP cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in RPMI-1640 media (GE Healthcare Life Sciences HyClone laboratories, Logan, UT, USA) with 10% fetal bovine serum (FBS, Clark Bioscience, Richmond, VA, USA).
To detect the effect of triptolide on cell growth, LNCaP were plated on 96-well plate (1.5 × 10⁵/well) and treated with triptolide (100 nM). The viable cells were detected with SRB assay [8 (link)].
To detect the AR protein level, after being plated on 6-well plates, LNCaP cells were pre-treated with CHX (20 ng/mL) for 2 h and then triptolide (100 nM) was added to the media. After co-treatment with CHX and triptolide for 24 h, the cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Haimen, Jiangsu, China) and the protein concentration was detected with BCA kit (Beyotime Biotechnology, Haimen, Jiangsu, China).
To detect the role of calpain in triptolide mediated AR degradation, calpain inhibitor ALLM and calcium chelator BAPTA-AM were used. After co-treatment with triptolide and ALLM (50 μM and 100 μM), PMSF (100 μM) or BAPTA-AM (10 μM) for 24 h, the cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Haimen, Jiangsu, China) and the protein concentration was detected with BCA kit (Beyotime Biotechnology, Haimen, Jiangsu, China).

Cells were lysed in RIPA Lysis Buffer (50 mM Tris pH 7.4, 120 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, Beyotime, China) supplemented with protease inhibitors (Complete Mini, Roche, Switzerland) and phosphatase inhibitors (PhosSTOP, Roche, Switzerland). Then, 1000 µg of total cell lysates were incubated with 1 µg of primary antibody or control rabbit IgG at 4 ℃ overnight. Then, 20 µL of Protein A + G agarose (Bioworld Technology, USA) was added for 2 h at 4 °C with rocking. The precipitates were washed four times with RIPA Lysis Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Beyotime, China).
Analysis was conducted on a Q Exactive mass spectrometer coupled to an Easy nLC (Thermo Fisher Scientific, USA) using a routine method. MS data were acquired using a data-dependent top 10 method dynamically selecting the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. The proteins identified from the blank control group were regarded as non-specific proteins and were removed from the protein list identified from the MCCC2 test group to exclude the non-specifically binding proteins of MCCC2.

HEK293Ta cells expressing HA-Inpp5e and Flag-OSBPL2 were washed 3 times with cold PBS and then lysed with RIPA lysis buffer (Beyotime) containing complete protease inhibitors (MCE) for 30 minutes. The supernatant was collected and incubated with anti-FLAG M2 magnetic beads (Sigma-Aldrich) overnight at 4°C. The beads were washed with RIPA lysis buffer (Beyotime) at least 5 times. Immobilized protein complexes were eluted by denaturation in 5 × SDS loading buffer at 100°C for 10 minutes and then assayed by Western blot.

Cells were lysed in RIPA lysis buffer (Beyotime, Beijing) according to the manufacturer's instructions. Protein extracts were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes (Roche, Switzerland). Membranes were blocked in 5% BSA/PBST at room temperature for one hour and incubated with primary antibodies overnight. Immunoblots were detected with the Tanon^TM High-sig ECL Western blotting substrate (Tanon, Shanghai). The information of the primary antibodies is listed in Table S2. For the coimmunoprecipitation assay, cell lysates were prepared using RIPA lysis buffer (Beyotime, Beijing). Whole cellular extracts were immunoprecipitated with ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, USA) (for exogenous binding) or anti-DDX21 antibody (for endogenous binding), or isotype IgG (as a control) (Cell Signaling, USA) with Protein A resin (GeneScript, Nanjing) overnight at 4 °C. Chromatin immunoprecipitation was performed as described previously³⁵. ChIP-primer sequences are listed in the Table S3.

The cells were lysed by ice-cold RIPA lysis buffer (Biyuntian, China) containing protease inhibitors, and the protein concentration was quantified using a BCA protein assay kit (Biyuntian). Each group of proteins was denatured by boiling, and then sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins. Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by electrophoresis. Then, the membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4 °C in diluted primary antibody. Finally, images of the target strip were developed using a Western Chemiluminescent HRP Substrate Kit (Millipore, USA). Image Lab software (Bio-Rad, USA) was used for semi-quantitative analysis. Primary antibodies directed against AMPK (D5A2 for monoclonal antibody, Cell Signaling Technology), p-AMPK (40H9 for monoclonal antibody, Cell Signaling Technology), β-catenin (polyclonal antibodies, Cell Signaling Technology), p-β-catenin (polyclonal antibodies, Cell Signaling Technology), p-Smad-1/5/8(D5B10 for monoclonal antibody, Cell Signaling Technology), Smad-1/5/8 (N-18 for monoclonal antibody, Santa Cruz Biotechnology) and GADPH (6C5 for monoclonal antibody, Beyotime) were used.