Ta 09

The extracted protein from 10% tissue homogenate was used to evaluate the expression level of cell cycle regulator, cyclin D1 and invasiveness-related proteins, MMP-2 and MMP-9. The total protein from colon tissues was separated by SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) electrophoresis and electrotransferred to a PVDF (Polyvinylidene Fluoride) membrane. The PVDF membranes were blocked using TBST, which contains 5% BSA for 2.5 h at room temperature. They were further incubated for 2 h with anti-cyclin D1 rabbit monoclonal antibody (#2978, 1:1000 dilution, Cell Signaling Technology, Tokyo, Japan), anti-MMP-2 goat polyclonal antibody (AF1488, 1:1000 dilution, R&D Systems, Minneapolis, MN, USA), anti-MMP-9 goat polyclonal antibody (AF909, 1:1000 dilution, R D Systems, Minneapolis, MN, USA) and anti-β-actin mouse monoclonal antibody (TA09, 1:2000 dilution, ZSGB-BIO, Beijing, China). After washing three times with TBST, the PVDF membranes were incubated with an HRP-conjugated secondary antibody (ZB-2306, ZB2301, 1:2000 dilution, ZSGB-BIO, Beijing, China) for 1.5 h at room temperature. The PVDF membrane was washed three times with TBST, and the immunoreactive protein was visualized by using the enhanced chemiluminescent reagent (Supersignal Chemiluminescent Substrate, Pierce, Rockford, IL, USA).

Human HMC3 microglial cells were stimulated with LPS (1 μg/mL) for 24 h, then cells were treated with 1 μM and 10 μM samples for 24 h. Cells were next lysed with Western blot cell lysis buffer (BioSharp, Hefei, China), added to the sample buffer, boiled and stored at −80 °C. Cell lysates were separated by SDS-PAGE (BioSharp, Hefei, China) and electrophoresed on PVDF membranes. The antibodies against IKK (#61294s, Cell Signaling Technology, Danvers, MA, USA), p-IKK (#2697s, Cell Signaling Technology, Danvers, MA, USA), IκBα (#4812s, Cell Signaling Technology, Danvers, MA, USA), p65 (#8242s, Cell Signaling Technology, Danvers, MA, USA), p-p65 (#3033s, Cell Signaling Technology, Danvers, MA, USA), and β-actin (TA-09, zsbio, Beijing, China) were closed with 5% skimmed milk powder in a shaker for 2 h, and washed three times with a TBST buffer at 4 °C, diluted with anti-rabbit (ZB-2301, zsbio, Beijing, China) or anti-mouse (ZB-2305, zsbio, Beijing, China) lgG horseradish enzyme in TBST (1:8000), and incubated at room temperature for 2 h. After treatment with chemiluminescence agent (BioSharp, Hefei, China), protein bands were detected by a chemiluminescence instrument (SAGECREATION, Beijing, China) and quantified by Image J 1.52a (National Institutes of Health, Bethesda, MD, USA) [40 (link)].

Human spermatozoa from AY078 and control fertile groups were washed three times with PBS, dissolved using 1×SDS loading buffer (Beyotime Biotechnology,China), and denatured at 100°C to avoid protein loss due to inadequate lysis. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:1000), rabbit polyclonal anti-SPAG6 (HPA038440, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:1000), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:1000), and mouse polyclonal anti-β-actin (TA-09, ZSGB-Bio, China). After incubation with secondary antibodies at 37°C for 2 h, blots were visualized and analyzed(Tanon 5200,China).

SMM103 cells (1000/well) were seeded into 6-well plate, after culturing for 24 h, cells were treated with various concentrations of sirolimus (0, 0.01, 0.1, 1, 10, 100 nM) for 72 h or treated with 100 nM sirolimus at various timepoints (0, 3, 6, 24, 48, 72 h). Afterward, the cells were washed with cold PBS and lysed in cold RIPA buffer (20-188, Merck Millipore) supplemented with protease inhibitor (Bimake, B14001) and phosphatase inhibitor (Bimake, B15002). Following antibodies were used: Calreticulin antibody (abcam, 1:1000, ab92516), HSP70 antibody (abcam, 1:1000, ab5439), phosphor-AKT (Ser 473) antibody (CST, 1:100, 4060L), phosphor-S6 (Ser240/244) antibody (CST, 1:1000, 5364s), beta-actin antibody (Zsbio, 1:2000, TA-09).

Total proteins were extracted from backfat using radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) and protein content was measured using the bicinchoninic acid protein assay kit (Beyotime, China). Total protein (5 to 20 μL) was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride membrane. Blots were blocked with 5% skim milk overnight at 4°C and blotted with specific primary antibodies for LEPR (1:300, bs-20498R, Bioss, Beijing, China) and β-actin (1:2,000, TA-09, ZSGB-BIO, Beijing, China). The bound primary antibodies were determined with a horseradish peroxidase conjugated secondary antibody goat anti-rabbit immunoglobulin G (IgG) (1:2,000, ZB2301, Zsbio, Beijing, China) and a horseradish peroxidase conjugated secondary antibody goat anti-mouse IgG (1:2,000, ZB2305, Zsbio, China), respectively. The data were analyzed using Image J software (GEL system, Beijing kcrx bio-company, Beijing, China). The relative expression of LEPR was calculated as the ration of optical density of LEPR to that of β-actin.

U251 and LN229 cell lines were treated with siHYAL2 for 48 h. Then, the glioma cells were lysed in RIPA solution on ice for 30 min. Next, total protein was extracted from glioma cell lines. 12.5% SDS‒PAGE gels were used to separate protein samples, and then the protein samples were transferred to PVDF membranes. After routinely blocking the membranes and incubating them with the primary and secondary antibodies, the GeneGnome XRQ Imaging System (Syngene, UK) was used to observe the immunoreactions. The primary antibodies were as follows: anti-HYAL2 (DF13080, Affinity); anti-CCND1 (26939-1-AP, Proteintech); anti-CCNB1 (28603-1-AP, Proteintech); anti-Bcl-2 (60178-1-Ig, Proteintech); anti-BAX (50599-2-Ig, Proteintech); and anti-β-actin (TA-09, ZSGB-BIO).