Kgp250

Testis tissue (about 150 mg; n = 12 for each group) was lysed with radio immunoprecipitation assay (RIPA) lysis buffer, which was supplemented with phenylmethanesulfonyl fluoride and phosphatase/protease inhibitors (cat. No. KGP250, KeyGEN BioTECH, Nanjing, China). The tissue was homogenized to extract total proteins in a homogenate device (Leica, Heidelberg, Germany) under cold conditions. After that, the levels of the secretion factor of Sertoli cells (SCs), such as glial cell line-derived neurotrophic factor (GDNF), stem cell factor (SCF) (SEA043Mu, MEA120Mu, Cloud-Clone Corp., Katy, TX, USA), transferrin (TRF) and androgen binding protein (ABP) (SU-B31079, SU-B35221, Mlbio, Shanghai, China), were measured by a commercial enzyme linked immunosorbent assay (ELISA) kit according to the manufacturers′ instructions. Moreover, the level of serum testosterone (T), which was mainly secreted by Leydig cells (LCs), was determined by an ELISA kit (E-EL-00720, Elabscience, Wuhan, China) using the same method as above.

The cells were lysed in equal volumes of ice-cold lysis buffer containing a protease inhibitor cocktail (Pierce Chemical Co.). Cell homogenates were boiled for 5 min in 5× Laemmli sample buffer. Total proteins were extracted with a Whole Protein Extraction kit (KGP250, KeyGEN BioTECH, China). Polyvinylidene difluoride membranes were then blocked in 5% fat-free milk or 5% BSA in TBS containing 0.05% Tween 20. Following overnight incubation at 4 °C with antibodies targeting TLR5 (ab13876, Abcam, UK), MyD88 (ab133739, Abcam, UK), p65 (ab32536, Abcam, UK), phosphorylated p65 (p-p65, ab86299, Abcam, UK) and GAPDH (ab181602, Abcam, UK), the membranes were incubated with HRP-conjugated secondary antibodies (KGAA35, KGAA37, KeyGEN BioTECH, China) at a 1:150,000 dilution for 1 h at room temperature and developed with a SuperSignal chemiluminescent detection system.

The RIPA lysis buffer containing protease inhibitors (# KGP250, KeyGEN BioTECH, Nanjing, China) was used to extract PCa cell protein following the standard protocol. Then, equal amounts of proteins in the cell lysates were separated by SDS/PAGE gels (4%–12%, Bio‐Rad) and electronically transferred onto polyvinylidene fluoride (PVDF, Millipore) membranes. The membranes were then blocked with 5% bovine serum albumin (BSA) and incubated overnight at 4°C with the following specific primary antibodies: N Cadherin Antibody (T55015, Abmart), Vimentin Antibody (T55134, Abmart), GAPDH Antibody (MA9166, Abmart), IL10 Antibody (TD6894, Abmart), p62/SQSTM1 Antibody (T55546, Abmart), Beclin 1 Antibody (T55092, Abmart), LC3A/B Antibody (TA5402, Abmart), STAT3 Antibody (T55292, Abmart), Phospho‐STAT3 (Y705) Antibody (T56566, Abmart), JAK2 Antibody (T55287, Abmart) and Phospho‐JAK2 (Y1007 + Y1008) Antibody (T56570, Abmart). Subsequently, horseradish peroxidase (HRP)‐conjugated secondary antibody was used to incubate the samples for 1 h at room temperature. The bands were visualized using the enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockford, IL, United States).

Total protein was extracted from RAW 264.7 cells using the same procedures as described in detail elsewhere (Santa et al., 2016 (link)). Briefly, raw cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo, 89901) containing proteinase inhibitors and phosphatase inhibitors (KeyGEN BioTECH, KGP250). The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, 23,225). Subsequently, the phenol–ethanol supernatant was mixed with isopropanol to isolate proteins. Equal amounts of protein and supernatants were separated by 12.5 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 or 0.20 μm-pore polyvinylidene fluoride membranes (Merck Millipore, IPVH00010 or ISEQ00005). Membranes were incubated overnight with antibodies against CD163 (Abcam, ab182422), CD206 (Abcam, ab64693), PDGF-BB (Santa Cruz, sc365805), MMP9 (Abcam, ab38898), and β-actin (Cell Signaling echnology, 2118). The membranes were then incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) (h + l) secondary antibody (1:5,000) for 1 h. Protein signals were detected using an enhanced chemiluminescence kit (Cat. WBKLS0500, Millipore), and Western blot bands were examined and analyzed with a chemiluminescence instrument (Guangzhou Ewell Bio Technology Co. Ltd., China).

According to the instructions, the total protein was extracted from the globus pallidus with a total protein extraction kit (KGP250; KeyGen Biotech, Nanjing, China), and a bicinchoninic assay (BCA) protein detection kit (KGPBCA; KeyGen Biotech, Nanjing, China) was used to quantify the protein concentrations. Target proteins were isolated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane. After cleaning with Tris-buffered saline with a Tween 20 (TBST) solution, the PVDF membranes were sealed in a blocking solution for 15 min. They were incubated overnight at 4 °C with anti-GAD67 (1:1000; Abcam, Cambridge, UK), anti-caspase-3 (1:1000; Abcam, Cambridge, UK) and anti-β-actin (1:2000; ABclonal, Wuhan, China) antibodies. The next day, the PVDF membranes were washed with TBST and incubated with the respective secondary antibodies for 1 h. Subsequently, each PVDF membrane was treated with an enhanced chemiluminescence (ECL) solution and exposed using the Bio-Rad ChemiDoc Touch System (Bio-Rad Laboratories, Hercules, CA, USA). Image analysis was performed using ImageJ software (Version 1.53; NIH, Bethesda, MD, USA).