Image system

The cellular proteins were purified from treated or control cells. Equal amounts of protein (10 μg) were loaded in each lane for NuPAGE 4–12% Bis-tris gel and then transferred to polyvinylidene difluoride membranes. The membranes were washed with blotting buffer (1× PBS containing 0.1% Tween20) and then blocked for 60 min in blotting buffer containing 10% low-fat powdered milk. Membranes were washed 3 times with blotting buffer, incubated at 4 °C overnight with primary antibody (1:1000) containing 5% low fat powdered milk and incubated with HRP conjugated secondary antibody (1:1000) at room temperature for 60 min. The blots were detected with Bio-Rad image system. The relative expression of proteins was normalized to a-Tubulin (cat.no: ab7291), and analyzed using Image J. ASAH2 (cat.no: ab170949), SGPP1 (cat.no: ab108435), SPHK1 (cat.no: ab109522), SPHK2 (cat.no: ab264042), SMPD2 (cat.no: ab131330), SMPD3 (cat.no: ab172193) were purchased from Abcam. SGPP2 (cat.no: PA5-42767) was purchased from Thermo Fisher.

MEC-2 cells were treated with or without 100 µM flu for 24 hrs and flu-resistant clonal cells were cultured in “maintaining” medium containing 100 µM flu. After treatment, cells (dead and alive) were harvested by centrifugation at 1,500 rpm for 2 mins, and the pellets were re-suspended in 0.5 ml of lysis buffer containing 5 mM Tris-HCl, pH 8.0, 20 mM EDTA, and 0.5% Triton X-100 and placed on ice for >60 mins. The samples were then centrifuged at 14,500 rpm for 20 mins, and the supernatant containing DNA cleavage products with the same amount of cellular proteins was precipitated by isopropyl alcohol for 15 hrs. The samples were centrifuged at 14,500 rpm for 20 mins, and the pellets were re-suspended in Tris-EDTA buffer with proteinase K and RNase A for 2–3 hrs at 37° C. DNA fragments were separated on a 1.2% agarose gel, visualized with ethidium bromide, and photographed using the Bio-Rad Image System.

Total protein from ovarian carcinoma cell lines was extracted using lysis buffer (KeyGEN BioTECH, China) containing phosphatase and protease inhibitors (KeyGEN BioTECH, China). Protein concentration was detected using BCA protein assay kit (KeyGEN BioTECH, China). Equal amount of protein (10 μg) was loaded onto 10% SDS-PAGE (PG112, Epizyme Biotech, China) and then transferred to PVDF membranes (Millipore, USA). All membranes were then blocked in TBST for 1 h with 5% BSA at room temperature and followed by primary antibodies at 4 °C overnight. Then membranes were incubated at room temperature for 2 h with HRP-conjugated secondary antibodies (701,051, Zen Bioscience, China; ZB-2301, ZSGB-BIO, China). Protein signals were visualized with a chemiluminescence kit (Millipore, USA) and an Image system (Bio-Rad, USA). GAPDH was used as the internal control. Primary antibodies were as follows: MTHFD2 (ab151447, 1:3000, Abcam, Cambridge, MA, USA), MOB1A (ab236969, 1:3000, Abcam, Cambridge, MA, USA) and GAPDH (cat.no. T0004; 1:5000; Affinity Biosciences).

The subventricular zone (SVZ) tissue was separated and homogenized to collect the protein samples at 3 days after hemorrhage. Equal amounts of protein samples (20 μg) were loaded on SDS-PAGE gels, electrophoresed, and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked and incubated overnight at 4°C with the following primary antibodies: anti-ZO-1 (1 : 1000; Abcam, USA), anti-NLRP3 (1 : 1000; Abcam, USA), anti-Caspase1 (1 : 1000; NOVUS, USA), anti-IL1beta (1 : 1000; GeneTex, USA), anti-Atg5 (1 : 1000; ZEN-BIO, China), anti-LC3B (1 : 1000; ZEN-BIO, China), anti-p62 (1 : 1000; ZEN-BIO, China), anti-pAMPK (1 : 1000; CST, USA), anti-AMPK (1 : 1000; CST, USA), anti-ULK1 (1 : 1000; CST, USA), anti-Beclin-1 (1 : 1000; CST, USA), and anti-β-actin (1 : 1000; CST, USA). Appropriate secondary antibodies (1 : 3000, CST; 1 : 5000, abcam) were selected to incubate with the membrane for 1 h at room temperature. The bands were probed with an ECL Plus chemiluminescence regent Kit (ThermoFisher, USA) and visualized with the image system (Bio-Rad, USA). Relative density of the protein immunoblot images was analyzed by Image J software (NIH, USA).

Aortic rings were homogenized with homogenization buffer (Beyotime Institute of Biotechnology). And western blot analysis was used to determine the protein expression level as previously described [3 (link)]. The images were captured by Image System (Bio-Rad) with an enhanced chemiluminescence detection kit (Millipore). Protein concentration was determined by BCA assay kit (Pierce).

Three–week–old cultured DRG neurons were used for BoNT⁄A treatment and TRPV1 antibody interference experiments.
WB was used to determine the expression of SNAP–25, SV2A and TRPV1. Cultured DRG neurons were harvested using RIPA buffer (NaCl, 150 mM; Tris–base, 50 mM; EDTA, 2 mM; SDS, 0.1%; and Triton X–100, 1%; with fresh proteinase inhibitor). Cell extracts were then sonicated briefly and centrifuged. Supernatants were used for protein concentration assays and stored at –20°C for WB assays. Twenty micrograms of total protein was loaded onto 12.5% pre–cast gels (Bio-Rad, USA) for SDS–PAGE and WB analysis. Anti–SNAP-25 (1:6000), anti–SV2A (1:500) and anti–TRPV1 (1:500) antibodies were used to probe specific proteins. The positive protein bands were developed using chemiluminescence and imaged with a Bio–Rad Image System.
The specificities of both BoNT⁄A and TRPV1 were tested prior to this study. Two processes were used to confirm antibody specificities; 1) different concentrations of the antibodies were pre-incubated with blocking peptides and 2) primary antibodies were eliminated as negative controls. Finally, anti-BoNT⁄A at 1:8000 for immunofluorescent staining and at 1:15000 for WB, anti–TRPV1 at 1:200 for immunofluorescent staining and at 1:500 for WB was chosen.