Chemiluminescence detection system

Total protein was extracted with RIPA buffer supplemented with protease inhibitors. Protein extracts were resolved on 10% SDS–PAGE gels, transferred onto PVDF membranes (Millipore, Billerica, MA, USA), as descripted previously [14 (link)]. The primary antibodies anti-rabbit Smad4 (Cell Signaling, Danvers, MA, USA) or GAPDH (Kang Cheng Biotechnology, Shanghai, China) were applied overnight. The dilutions of the primary antibodies were 1:1000 for anti-SMAD4 and 1:10000 for anti-GAPDH. The following day, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies (Kang Cheng Biotechnology, Shanghai, China) at room temperature for 60 minutes. A chemiluminescence detection system (Millipore, Billerica, MA, USA) was used for visualization of the results.

BMMs were lysed in a lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitors. The lysate was centrifuged at 14,000 rpm for 20 min to obtain pure protein. The protein concentration was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA), and equal amounts of proteins were separated with a sodium dodecyl sulphate-polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) and treated with 5% nonfat dry milk to inhibit attachment of nonspecific proteins. After the membrane was treated with primary and secondary antibodies (horseradish peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit immunoglobulin), expression of specific protein signals was measured using a chemiluminescence detection system (Millipore).

The samples were prepared as previously described [15 (link)]. Ipsilateral and contralateral testes were lysed and centrifuged at 4°C (10,000 g, 10 minutes). The bicinchoninic acid (BCA) method was used to determine the protein concentration (Pierce, Rockford, IL USA). Equal amounts of protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide (SDS‒PAGE) gel electrophoresis and transferred onto nitrocellulose membranes (Pall Corporation, Pensacola, FL, USA). The immunoblots were incubated overnight at 4°C with primary antibodies against phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204) (p-ERK) and total ERK1/2 (1:1000, Cell Signaling, Danvers, MA, USA). Then, the membranes were incubated with the secondary anti-rabbit antibody (1:5000, Bio-Rad, Hercules, CA, USA) at room temperature for 1 hour. Signals were detected using a chemiluminescence detection system (Millipore, Billerica, MA, USA) on an Amersham Imager (600, GE Healthcare, Little Chalfont, UK). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the gray value of the band. The densities of phosphorylated bands were normalized to the total protein.

4T1 cells (1 × 10⁶ cells/100 mm culture dish) were plated in DMEM containing 100 mL/L FBS. The cells were then serum-starved for 24 h in DMEM and treated with or without various concentrations of HEGU or licoricidin for 18 h. Conditioned media was collected and concentrated as previously described [10 (link)]. Proteins in total cell lysates (50 μg protein) [36 (link)] and concentrated conditioned media (50 or 80 μg protein) [37 (link)] were subjected to Western blot analyses. The signal was detected with a chemiluminescence detection system (Millipore, Billerica, MA, USA). The relative abundance of each band was quantified using the Bio-profile Bio-ID application (Vilber-Lourmat, Marine La Vallee, France) and the control (0 μg/mL HEGU or licoricidin) levels were set to 1.

Proteins were extracted using a commercial kit (No. C510003, Sangon Biotech, China) according to the manufacturer’s instructions. The following primary antibodies were used (all at a 1:1,000 dilution): rabbit anti-RUNX2 (ab23981, Abcam), rabbit anti-OSX (ab94744, Abcam), rabbit anti-OCN (23418-1-AP, ProteinTech, Wuhan, China), rabbit anti-ALP (ab83259, Abcam), rabbit anti-ACP5 (ab191406, Abcam), rabbit anti-ITBG3BP (ab192324, Abcam), rabbit anti-MMP9 (10375-2-AP, ProteinTech, Wuhan, China), rabbit anti-CTSK (ab19027, Abcam), rabbit anti-GAPDH (10494-1-AP, ProteinTech, Wuhan, China), rabbit anti-FOXO3 (10849-1-AP, ProteinTech, Wuhan, China) and rabbit anti-TAK1 (ab109526, Abcam). The protein samples were separated by 10% SDS–PAGE and subsequently transferred to nitrocellulose filter membranes (Pall Corp., Washington, NY) using a wet transfer blotting system (Bio-Rad, Hercules, CA). After incubation, a goat anti-rabbit-HRP secondary antibody (Pierce, USA) was applied at a 1:2,000 dilution. The proteins were then detected by a chemiluminescence detection system (Millipore, USA). Anti-GAPDH was used as an endogenous control. The uncropped scans of all blots were provided in the Source Data file.

Parasite nuclear protein extract was prepared by high salt. The nuclear extract was further diluted, pre-cleared and incubated with RNAPII antibodies overnight at 4°C. Protein complexes were precipitated by incubating with Protein G agarose slurry for 1 h at 4°C followed by extensive washings to reduce the background. Protein complexes were eluted by addition of SDS-PAGE loading buffer and boiling for 5 min at 95°C. Supernatants were resolved in 6% SDS-PAGE and subsequently used for Western blot analysis. Western blots were carried out using antibodies raised against the CTD of RNAPII described in this study and the anti-CTD antibody 8WG16 (Covance) [39 (link)]. Secondary antibody (anti-mouse-HRP, Sigma) was visualized using a chemiluminescence detection system (Millipore) as per the manufacturer’s instructions.