α tubulin

Western blot assay was performed using the standard method as briefly described 21 (link),22 (link). Protein samples were collected after cells were lysed and quantified. 20~30 μg/lane protein were separated by 10%~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A wet transfer (Bio-Rad, USA) were then performed to transfer the protein onto polyvinylidene fluoride (PVDF) membranes and blocked with non-fat milk for 1 h. The membranes were incubated with primary antibody MFAP5 (HPA010553,Sigma,Germany), MMP9 (D6O3H, Cell signaling technology, USA), MMP2 (D4M2N Cell signaling technology, USA), vimentin (D21H3, Cell signaling technology, USA), Sanil (C15D3, Cell signaling technology, USA), AKT (11E7, Cell signaling technology, USA), p-AKT (D9E, Cell signaling technology, USA), HIF-1α (D1S7W, Cell signaling technology, USA), α-tubulin (11224, Proteintech, USA), β-actin (20536 Proteintech, USA) at 1:1000 dilution overnight and then with anti-rabbit HRP-linked IgG secondary antibody(7074, Cell signaling technology, USA) at 1:2000 dilution for 1 hour. The immunoreactive bands were visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China). α-tubulin and β-actin was used as an internal control.

Cells were lysed by shaking in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor (Nanjing Key Gen Biotech Co., Ltd., Nanjing, China). The protein concentration was measured by the bicinchoninic acid (BCA) assay (Beijing Trans Gen Biotech Co., Ltd. Beijing, China). Cell extract (50 μg) was applied to a sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, and then blocked with PBST containing 5% skimmed milk for 2 h. The membrane was incubated at 4 °C with primary antibodies (TLR4, 1:500; Myd88, 1:1000; iNOS, 1:1000; CD206, 1:2000; Arg-1, 1:1000 [Abcam]; IKK-β, 1:500; p-IKK-β, 1:200; IκBα, 1:1000; p-IκBα, 1:1000; NF-κB, 1:1000; p-NF-κB, 1:1000; GAPDH, 1:2000; α-tubulin, 1:1000; and β-tubulin, 1:1000 [Proteintech]) overnight. On the next day, the membrane was washed four times for 8 min each with PBST and incubated with HRP-anti-rabbit secondary antibody (1:5000; Proteintech) for 1 h. The bands were visualised by enhanced chemiluminescence kit (Advansta, Menlo Park, CA, USA), and the results were analysed using Quantity One (Bio-Rad Laboratories, Inc. Hercules, CA, USA) software. GAPDH, α-tubulin, and β-tubulin were used as internal references.

Total protein of testes was extracted with the Total Protein Extraction Kit (Invent) and the protein concentration was examined by BCA method (Thermo). Protein samples were separated by SDS-PAGE and then transferred to PVDF membrane (Millipore). After blocking in 5% milk, the membranes were incubated with indicated primary antibodies at 4 °C overnight. The following antibodies were used: FANCI (Novus, 1:5000), FLAG (Sigma, 1:5000), and α-Tubulin (Proteintech, 1:3000). After being incubated with secondary antibodies, the membranes were detected with an ECL system (Millipore). The ChemiDoc MP System (Bio-Rad) was used to capture images.

Antibodies against USP7 (Cat. CST#4833), K48Ub (Cat. CST#4289), and K63Ub (Cat. CST#12930) were purchased from Cell Signaling Technologies, Inc. The antibodies against RNF6 (Cat. #20437-1-AP), USP9x (Cat. #55054-1-AP), PARP (Cat. #13371-1-AP), Caspase 3 (Cat. #19677-1-AP), GAPDH (Cat. #60004-1-Ig), α-tubulin (Cat. #11224-1-AP), and V5 (Cat. #14440-1-AP) were obtained from Proteintech. The monoclonal antibodies including anti-Flag (Cat. #M185-3L), anti-HA (Cat. #M180-3), and anti-Myc (Cat. #M192-3) were obtained from Medical and Biological Laboratories Co Ltd. The anti-Ub antibody (Cat. #SL-8017) was purchased from Santa Cruz Biotechnology, Inc. HRP-labeled goat anti-mouse (Cat. #A0216) and goat anti-rabbit IgG (H + L) antibodies (Cat. #A0208) were purchased from Beyotime Institute of Biotechnology. MG132 (Cat. #S2619), Bortezomib (Cat. #S1013), and P5091 (Cat. #S7132) were purchased from Santa Cruz Biotechnology and Selleck Chemicals Inc, respectively. CHX (Cat. #C7698) and chloroquine (CHQ, Cat. #C6628) were purchased from Sigma-Aldrich. LBH589 (LBH, Cat. #M1748) was purchased from AbMole Bioscience Inc. Nilotinib (Cat. #IN0560) was purchased from Solarbio Life Science. Recombinant ubiquitin (Cat. #U100-H), UBE1 (Cat. #E-304), UBE2D1(Cat. #E2-616), and ATP (Cat. #B-20) were all purchased from Boston Biochem Inc.