Sds page protein loading buffer

Nobiletin (Nob, IN0210) was obtained from MedChemExpress (Beijing, China). Nob was prepared as a stock solution with DMSO, dispense and kept at −80 °C and use up within six months. D-galactose (D-gal, G5388) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA Lysis Buffer (P0013B), Phenylmethanesulfonyl fluoride (PMSF, ST506), 5× SDS-PAGE protein loading buffer (P0015L), BCA Protein Assay Kit (P0012), Mouse TNF-α ELISA Kit (PT512), and Mouse IL-6 ELISA Kit (PI326) were purchased from Beyotime Biotechnology Company (Shanghai, China). cDNA synthesis kit (G592) was obtained from abm (Vancouver, Canada). PowerUp™ SYBR™ Green (A25742) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Mouse LBP ELISA Kit (ab269542) was obtained from abcam (Shanghai, China). The primers were purchased from Beijing Tianyi Huiyuan Biotechnology Co., Ltd. (Beinjing, China). p-Ser2448-mTOR (5536T), p-p70 S6 Kinase Antibody (9234S) and p70 S6 Kinase Antibody (9202S) were purchased from Cell Signaling Technology. Furthermore, mToR (28273-1-AP) and GAPDH Polyclonal antibody (10494-1-AP) were obtained from Proteintech.

Phosphate-buffered saline (PBS) and Dulbecco’s modified Eagle’s medium (DMEM) were procured from Hyclone (United States). Type II collagenase and fetal bovine serum (FBS) were procured from Gibco (United States). 1% penicillin/streptomycin solution (100×), methyl thiazolyl tetrazolium (MTT), dimethyl sulfoxide (DMSO), NO synthase detection kit, NO detection kit, RIPA cell lysis buffer, phenylmethanesulfonyl fluoride (PMSF), and 5 × SDS-PAGE protein loading buffer were supplied by Beyotime (China). Recombinant rat IL-1β was purchased from Peprotech (United States). Antibodies against iNOS (ab178945), collagen type II (COL2A1; ab34712), matrix metalloproteinase 3 (MMP3; ab52915), cyclooxygenase-2 (COX-2; ab179800), matrix metalloproteinase 13 (MMP13; ab39012), and iNOS inhibitor 1400W (ab120165) were supplied by Abcam (United States). Antibodies against aggrecan (ACAN; DF7561), glyceraldehyde phosphate dehydrogenase (GAPDH; AF7021), Goat anti-rabbit IgG (H + L) HRP (S0001), and Goat anti-rabbit IgG (H + L) FITC (S0008) were procured from Affinity (China).

CAB cells (or EPC cells) were seeded in 10 cm² dishes overnight and then transfected with a total of 10 μg of various plasmid combinations. The transfected cells were washed twice with 10 mL ice-cold PBS and then lysed by radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). After removing cellular debris, the supernatant was transferred to a 1.5 mL clean tube and incubated with 25 μL anti-Flag Affinity gel (Sigma-Aldrich) or anti-HA Magnetic Beads (Thermo Fisher) overnight at 4°C with constant rotating incubation. Immunoprecipitated proteins were collected by Magnetic Stand (Promega), washed five times with lysis buffer, and resuspended in 100 μL SDS-PAGE protein loading buffer (Beyotime). The immunoprecipitates and whole cell lysates (WCLs) were separated by 10–12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore) for subsequent western blot analysis. Antibodies were diluted as follows: anti-β-actin (Cell Signaling Technology) at 1:3,000, anti-Flag/HA antibody (Cell Signaling Technology) at 1:3,000, anti-Myc antibody (Abcam) at 1:2,000, and HRP-conjugated anti-rabbit IgG (Thermo Scientific) at 1:5,000. Images were captured by ImageQuant LAS 4000mini (GE). Results were representative of three independent experiments.

The ovaries from NS, HSA, and hAMSC groups were pooled separately in SDS-PAGE protein loading buffer (Beyotime, P0015) and RIPA Lysis and Extraction Buffer (Thermo Fisher, 89900) with protease and phosphatase inhibitor Cocktail (Thermo Fisher, 78443). Then samples were denatured at 95 °C for 10 min and stored at − 80 °C. After thawing, they are loaded on a 10% SDS-PAGE and blotted on polyvinyl fluoride (PVDF) membranes (Bio-Rad, 1620177). Non-specific binding sites were blocked for 1 h at 37 °C with 5% no-fat dry milk (BD, 2321000) in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). Membranes were incubated with polyclonal rabbit anti-Sod1 (1:1000, Abcam, ab13498) , rabbit anti-FoxO3a (1:1000, CST, 12829), rabbit anti-p-FoxO3a (1:1000, CST, 9466), rabbit anti-Ampk (1:1000, Abcam, ab133448), rabbit anti-FSHR (1:1000, Proteintech, 22665-1-AP), rabbit anti-Cyp17a1(1:1000, ABGENT, AP7879c), and rabbit anti-GAPDH (1:5000, Proteintech, 10494-1-AP) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody (1:1000, Abcam,ab6721) for 1 h at room temperature. After washing, specific immunoreactive complexes were detected by ECL kit (Thermo Fisher, 32209). The bands were normalized for Gapdh using ImageJ software (NIH, Bethesda, MD, USA), and values were given as relative units. The experiment was performed in triplicate.

Total proteins in spleens or aortas were extracted via High Efficiency RIPA Lysis Buffer (KeyGEN BioTECH), quantified by BCA protein concentration assay kit (Beyotime Biotechnology), and denatured in boiling water with SDS-PAGE protein loading buffer (Beyotime Biotechnology). 30 μg of proteins per sample was loaded in 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as blocking agent before antibody incubation. Polyvinylidene fluoride (PVDF) membranes were incubated with rabbit antibodies against p-STAT3-Y705 (Abcam), p-STAT5-Y694 (Cell Signaling Technology), SOCS3 (Abcam), FOXP3 (Abcam), β-ACTIN (Abways), and Goat anti-rabbit IgG with HRP (Biosharp), respectively. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Blot data were analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas are measured, and the relative expression amount of the protein samples is calculated by the method of the target protein gray value/internal reference β-ACTIN gray value.

Western blotting was performed to detect the expression of HMGB1 in the supernatant of PC-3 cells after 24 h of drug treatment (DOX: 3 µg/mL, BP-P: 40 µg/mL, BP-P+ laser: 40 µg/mL, BP-P-Apt-Zn/DOX: 40 µg/mL, and BP-P-Apt-Zn/DOX + laser: 40 µg/mL). The procedure was as follows: β-actin was used as the control protein. The supernatant was collected after 24 h of drug treatment and centrifuged at 12000 rpm for 15 min to remove cell debris. The samples were then mixed with SDS-PAGE protein loading buffer (Beyotime, China) and boiled for 5 min. About 20 µL of the samples were subjected to SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with rabbit monoclonal antibody (1:1000, CST, Boston, USA) and gently shaken at 4°C overnight. After 3 washes, the membranes were incubated with anti-Rabbit IgG and HRP-Linked Antibodies (1:2000, CST, Boston, USA) for 1 h at room temperature. After three washes, the HMGB1 protein bands were detected using Bio-RAD (California, USA), and the protein concentration was analyzed using ImageJ.

Co-IP and western blotting were performed as described previously (62 (link)). Briefly, the CAB or 293T cells were seeded into 10 cm² dishes overnight, and transfected with different plasmids (a total of 10 μg) indicated on the Figure using FuGENE HD Transfection Reagent (Promega). At 24 h post-transfection, the cells were lysed by radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). After removing cellular debris, the supernatants were incubated with 15 μl anti-Flag or anti-Myc affinity gel (Sigma-Aldrich) overnight at 4°C. Immunoprecipitated proteins were resuspended in 100 μl SDS-PAGE protein loading buffer (Beyotime) after collecting by centrifugation and washing three times with lysis buffer. The whole cell lysates (WCL) or immunoprecipitated proteins were separated by 10–15% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Bio-Rad). The membranes were incubated with anti-β-actin (Cell Signaling Technology) at 1:3,000, anti-Flag/HA (Sigma-Aldrich) at 1:3,000, or anti-Myc (Santa Cruz Biotechnology) at 1:3,000, and then hybridized with the secondary HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Thermo Scientific) at 1:5,000. Results are captured by using an ImageQuant LAS 4000 system (GE Healthcare) and are representative of three independent experiments.