Goat anti rabbit hrp

Total protein was extracted from transfected cells using RIPA buffer (Beyotime, China) containing a protease inhibitor cocktail (Roche, USA). Protein concentration was quantified by a BCA protein assay kit (Beyotime, China). Equal amounts of protein in each sample were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). After blocking the membranes with 5% nonfat milk, primary antibodies were added and incubated for 1 h. The primary antibodies for Western blotting were as follows: anti-PD-L1 (CUSABIO, CSB-MA878942A1m), anti-PI3K (Cell Signaling Technology, 4249S), anti-AKT (HUABIO, ET1609-51), anti-p-AKT (HUABIO, ET1607-73) and anti-GAPDH (GENE TEX, GTX100118). After washing step, the membrane was incubated with secondary antibodies (goat anti-mouse HRP (Beyotime, A0563) or goat anti-rabbit HRP (Beyotime, A0516). Then, an ECL chromogenic substrate (BIO-RAD, USA) was applied for detecting the signals.

Antibodies for Apaf-1, CHOP, TRADD, Bcl-2 and Bax were purchased from Santa (Dallas, USA). Antibodies for Endo-G, Cyt-c, AIF, GRP78, Caspase3, Caspase4, Caspase8 and Caspase-9 were purchased from Abcam (Cambridge, USA). Antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technologies (Danvers, USA). Secondary antibodies of goat anti-mouse FITC, goat anti-rabbit HRP and goat anti-mouse HRP were purchased from the Beyotime Institute of Technology(Shanghai, China).

The tissues of the soleus muscles were extracted and lysed using RIPA buffer (Beyotime, China). The protein concentration was the determined using BCA method, followed by boiling and centrifugation. For SDS-PAGE analysis, 30 ug of protein was loaded and separated. The resulting protein was then transferred to PVDF membranes (Millipore, USA) and blocked with 5% non-fat dry milk for one hour at room temperature. After washing with PBST, the membranes were incubated with primary antibodies against NFATC2 (1:1000, CST, USA), CaN (1:1000, CST, USA), or GAPDH (1:500, Santa Cruz, USA) at 4 °C overnight, and subsequently incubated with goat anti-rabbit-HRP (1:2000, Beyotime, China) secondary antibodies at room temperature for 1 h. The presence of immunoreactive bands was detected using ChemiDoc™ Touch Imaging System (Bio-Rad, USA).

In this study, the A2058 and SKMEL28 cells were lysed with RIPA lysis buffer (Beyotime, Jiangsu, China) at 4 °C for 30 min after the cells were transfected, which contain protease inhibitors (Roche, Indianapolis, IN, USA), and the BCA kit (Beyotime) was used to measure the protein concentration. Ten lane-volumes of proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (0.22 µm; Millipore, Billerica, MA, USA). The membranes blocked with 5% nonfat milk (LP0033; Oxoid, Basingstoke, Hampshire, UK) for 1 h at room temperature, and incubated the membrane with relevant antibodies overnight at 4 °C: against METTL7A (1:1000, GTX65969; Genetex, Irvine, CA, USA), THBS1 (1:1000, 18304-1-AP; Proteintech, Rosemont, IL, USA), p53 (1:1000, 250143; ZenBio, Durham, NC, USA), p21 (1:1000, 2947; CST, Danvers, MA, USA), Cyclin D1 (1:1000, CST, 55506) and GAPDH (1:1000, GTX100118; Genetex). All antibodies were diluted with 5% Bovine Serum Albumin (WXBC4157V; Vetec, Hohenhameln, Germany). After being washed, the membrane was incubated with secondary antibodies (goat anti-mouse HRP (1:3000, A0563; Beyotime) or goat anti-rabbit HRP (1:3000, A0516; Beyotime). Secondary antibodies are diluted with 5% nonfat milk. A chromogenic substrate (Bio-Rad, Hercules, CA, USA) such as ECL is used for visualization.

Reagents used in this study were BBR and DDP (Sigma, St. Louis, MO, USA); antibodies for Ki67, PCNA, Caspase3, Clv C3, Caspase8, Clv C8, RIPK3, p-RIPK3 and p-MLKL (Abcam, Cambridge, MA, USA); antibodies for Cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technologies, Danvers, MA, USA); antibody for MLKL (Novus Biologicals, Littleton, CO, USA); and secondary antibodies of donkey anti-goat HRP, goat anti-rabbit HRP and goat anti-mouse HRP (Beyotime Institute of Technology, China).

To confirm the success of EX isolation, ~ 30–40 μg of exosomal protein was loaded onto a polyacrylamide gel (5% stacking and 10% resolving gel). After running the gel at 90 V for 20 min, followed by 120 V for 60 min in running buffer, the proteins were transferred to a polyvinyl fluoride membrane (0.45 μm; Millipore) and blocked with 5% skim milk (BD, Lincoln, NE, USA) for 2 h. The membranes were incubated overnight with primary antibodies (Abs) against CD63, CD9, C81 (rabbit pAb; SBI, Palo Alto, CA, USA), pregnancy-specific globin (PSG1), nestin (NES), progesterone receptor [PGR] (rabbit pAb; Absin, Shanghai, China), L1 cell adhesion molecule [L1CAM] (rabbit pAb; Abcam, Cambridge, UK), and placental alkaline phosphatase [PLAP](mouse mAb; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4 °C. The Ab dilution ranged from 500- to 1000-fold times, as appropriate. The next day, the membranes were washed and incubated with secondary antibodies (goat anti-rabbit-HRP; Beyotime, Shanghai, China). The blotted proteins were visualized using an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA), and the signal was detected using Saga (Sage Sciences, Lincoln, NE, USA).