Protein loading buffer

Co-IP assays were performed as described by Zhang et al. (2014 (link)). The transfected 293 T cells were harvested and lysed with lysis buffer (1 M Tris-HCl, pH 7.4, 5 M NaCl, 0.5 M EDTA, 1% Triton X-100, and protease inhibitors). The cell lysates were centrifuged at 12,000 rpm for 5 min, and the supernatant was collected and incubated overnight with anti-FLAG M2 affinity antibody (Sigma-Aldrich, A2220) at 4°C and washed 4 times with ice-cold lysis buffer. The samples were eluted with protein loading buffer (Solarbio, P1040) and subjected to SDS-PAGE and Western blotting with anti-Myc antibody (1:1,000) or anti-Flag tag antibody (1:1,000).

The LMH cells were seeded in 12-well plate the day before infection. The recombinant virus with 0.1 MOI was then inoculated into the LMH cells. At 48 hpi, the LMH cells and supernatants were harvested, the cells were then washed once with PBS buffer, and subsequently lysed in 100 μl lysis buffer with proteolytic protease and phosphatase inhibitor cocktail (NCM, Soochow, China). The lysates were then centrifuged at 12,000 rpm for 10 min to remove debris, the supernatants of lysates or supernatants collected from the 12-well plate were then boiled with protein loading buffer (Solarbio, Beijing, China). The samples were separated on a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes (Cytiva, MA, USA). The membranes were then blocked with NCMblot blocking buffer (NCM, Soochow, China) for 30 min at room temperature (RT), and incubated with corresponding antibodies. After being washed with PBST for three times, the membranes were then incubated with HRP-labeled secondary antibodies for 1 h at RT. After another three washes, the membranes were developed with chemiluminescent regents and imaged with an automatic imaging system (Tanon 5200).

Trizol and HPLC-grade acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, United States). LB was supplied by National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Pterostilbene (used as the IS, Figure 1B) was purchased from Great Forest Biomedical Ltd. (Hangzhou, China). NADPH-regenerating solution was obtained from iPhase Pharmaceutical Services (Beijing, China, containing 21.7 mM of NADP⁺, 55 mM of G-6-P, 6.67 U/mL of G-6-PDH and 55 mM of MgCl₂). Cimetidine, α-naphthoflavone, quinindium, 4-methylpyrazole, and ketoconazole were from J&K Chemical (Beijing, China). Oligo(dT) 15 primer, RNase-free water, RNasin Plus RNase Inhibitor, 5× M-MLV Reverse Transcriptase buffer, M-MLV Reverse Transcriptase and dNTPs were obtained from Promega Corporation (Madison, WI, United States). SYBR^® Premix Ex Taq™ (Tli RNaseH Plus) was from TAKARA BIO INC. (Dalian, China). Enhanced RIPA lysis buffer and protein loading buffer were purchased from Solarbio Life Sciences (Beijing, China). Primary antibodies of CYP1A2, CYP2C11, CYP2D1, CYP2E1, and CYP3A2 were supplied by Abcam PLC (Cambridge, United Kingdom). Primary antibody of GAPDH was from Beyotime Biotechnology (Shanghai, China) and HRP-conjugated secondary antibody was from ComWin Biotech Co., Ltd. (Beijing, China). All other chemicals and solvents were of analytical grade.

Primary antibodies including IκB-α, p-IκBα, and NF-κB-p65 were purchased from Cell Signaling Technology (Danvers, MA, USA). Nrf-2, HO-1, and β-actin were purchased from Cambridge Bio (Boston, MA, USA). The NanoDrop 2000 Ultra Micro Spectrophotometer was purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Gallic acid, sodium nitrite, the Prime Script TM RT Master Mix kit and TB Green^® Premix Ex Taq TM were purchased from Biotech Co. Ltd. (Shanghai, China). Protein loading buffer, 20% SDS, electroporation solution and Tris-HCl buffer were purchased from Solarbio (Beijing, China). BCIP/NBT alkaline phosphatase developer and 40% Acr-Bis were purchased from Beyotime Biotechnology (Shanghai, China). The CCK-8 kit was purchased from Dojindo Laboratories (Kumamoto, Japan). Amberlite XAD-2 resin was purchased from Sigma-Aldrich Trading Co. Ltd. (Shanghai, China). Other chemicals, including the LPS (Escherichia coli 0127: B8) and alkaline phosphatase-conjugated secondary antibody (anti-rabbit IgG) and the standards applied in the chemical analysis were purchased from Sigma (St. Louis, MO, USA).

Goat ovarian tissues were incubated on ice for 30 min in 1.5 mL centrifuge tubes containing lysis buffer, and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was collected and stored at −80 °C. The protein concentration was measured with a BCA assay kit (Solarbio, Beijing, China), and then 30 μg of protein was mixed with protein loading buffer (Solarbio, Beijing, China) and boiled at 99 °C for 10 min. The protein samples were then subjected to gel electrophoresis at 100 V on a 10% Bis-Tris gel. The proteins were transferred onto a nitrocellulose membrane and blocked at 4 °C overnight. Anti-PPP2R5C and anti-SLC39A5 were purchased from Proteintech company (Proteintech, Wuhan, China). All primary antibodies were diluted at 1:10,000 with primary antibody diluent (P0256-500 mL, Bain-marie, China) and washed three times with TBST before being washed with HRP-conjugated anti-rabbit IgG (Proteintech, Wuhan, China) with secondary antibody dilution (P0258-500 mL, Baymax, China) diluted to 1:5000 used to incubate the membrane for 2 h at room temperature. Western blots were visualized on an Odyssey CLX imaging system (Li-COR) (Bio-Rad, Hercules, CA, USA) with Supersignal HRP chemiluminescent substrate (Beyotime, Beijing, China) according to the manufacturer’s instructions.