Chemiluminescence reagent

Western blot was performed as previously described.²² Total proteins (20 μg) were subjected to SDS‐PAGE and then transferred onto a polyvinyl indene difluoride membrane (PVDF; Millipore, Billerica, MA, USA). Nonspecific binding was blocked with PBST (0.5% Tween 20 in PBS) containing 5% non‐fat milk (Shandong Sparkjade Biotechnology Co., Ltd.) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with individual primary antibodies in PBST containing 1% non‐fat milk (mouse anti‐IL‐6, rabbit anti‐TNF‐α, rabbit anti‐IL‐1β, rabbit anti‐GFAP, 1:1000, Abcam; mouse anti‐GFAP, rabbit anti‐pSTAT3, mouse anti‐STAT3, rabbit anti‐JAK‐1, rabbit anti‐JAK‐2, 1:1000, Cell Signaling Technology, rabbit anti‐β‐actin, Biosynthesis Biotechnology Inc., Beijing, China). Following three washes with PBST, the membranes were then incubated with the secondary antibodies (HRP‐linked anti‐rabbit IgG; HRP‐linked anti‐mouse IgG; 1:2000; Cell Signaling Technology). Then, the proteins were detected by chemiluminescence reagents (Millipore) and observed using a ChemiDoc™ XRS+ Imaging System (Bio‐RAD, Hercules, USA). The protein levels were quantified by densitometry using Image J 1.4.3.67.

The pretreated HUVECs were washed by pre-cooling PBS three times, and the proteins were extracted by RIPA lysis containing 1% PMSF (Solarbio, Beijing, China). The concentration of proteins was measured according to the BCA protein assay kit (Solarbio, Beijing, China). The proteins were boiled for 5 min in SDS-PAGE loading buffer, and 30 μg of proteins was loaded to each lane and separated by 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were blocked by 5% non-fat milk for 1 h and then immersed in primary antibodies overnight at 4 °C; the primary antibodies are as follows: rabbit anti-GAPDH (1:10000; Proteintech, Chicago, IN, USA), rabbit anti-PLGF (1:1000; Abcam, Cambridge, UK), rabbit anti-SCF (1:10000; Abcam), and rabbit anti-VEGFR2 (1:1000; Cell Signaling Technology, USA). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000; Proteintech) for 1 h. Chemiluminescence reagents (Millipore) were used for the development of band visualization. ImageJ software was used to analyzed the protein expression levels.

The cells were suspended in cold lysis buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 420 mM NaCl, 10% glycerol, 0.5% NP-40 containing proteases inhibitor, and 1 mM DTT) and incubated on ice for 15 min. After that, the cell lysates were hardly centrifuged for 10 min at 4 °C, and the supernatants were collected and boiled in protein sample buffer (0.7 M β-mercaptoethanol, 4% SDS, 0.17 M Tris, pH 6.8, 0.17 mg/mL bromophenol blue, and 10% glycerol). The boiled samples were analyzed by SDS polyacrylamide gels. The resolved proteins were transferred onto the nitrocellulose membrane. The membranes were blocked in 1% milk in PBST buffer (PBS with 0.05% Tween 20) for 30 min and incubated with primary antibody for 2 h at RT. The membranes were washed three times by PBST buffer with 0.5% milk and then incubated with appropriate HRP-linked secondary antibody for 1 h at RT. After washing, the bound antibodies were detected by chemiluminescence reagents (Millipore, Burlington, MA, USA). The primary antibodies used are as follows: PARP (#9542; Cell signaling, Danvers, MA, USA), caspase 3 (#9662; Cell signaling), GRP78 (#3177; Cell signaling), IRE1a (#3294; Cell signaling), PERK (#5683; Cell signaling), CHOP (#2895; Cell signaling), GAPDH (#2118; Cell signaling), and beta-actin (NB600-501, Novus Biologicals, Littleton, CO, USA).

Cells were collected after 5% trypsinization of the culture plate, followed by centrifuging at 12,000 rpm at 4 °C for 10 min. Cell pellets were resuspended in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor sodium orthovanadate (Santa Cruz Biotechnology Inc., Dallas, TX, USA) on ice for 30 min with vortex every 10 min. Protein concentrations were measured using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). An amount of 20 µg proteins from each sample were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (PerkinElmer, Waltham, MA, USA). Membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich). To detect target proteins, membranes were probed with specific primary antibodies overnight. The next day, membranes were washed for 10 min with TBS-T 3 times, followed by incubation with anti-rabbit or anti-mouse secondary antibodies. Protein bands were detected using chemiluminescence reagents (Millipore, Billerica, MA, USA).

Equal quantities of protein were analysed via 8 to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly (vinylidene difluoride) membranes. After incubation at 4 °C with primary antibodies against MMP1 (Proteintech Group, Rosemont, IL, USA), p-p38, p38, Bax, Cytochrome c and GAPDH (Cell Signalling Technologies, Boston, MA, USA) overnight, the blots were washed, exposed to corresponding HRP-conjugated secondary antibodies for 1 h, and finally detected by chemiluminescence reagents (Millipore, Billerica, MA, USA).

The mixture of protease and phosphatase inhibitors (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM NaF, 1 mM Na₃VO₄, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 lg/ml aprotinin and 1 lg/ml leupeptin) were useing for protein extracts. Extracted protein should be centrifugated at 14,000 g to remove debris. Denatured proteins in the samples were subjected to 10% SDS-PAGE. Anti-CK2α polyclonal antibody (1:1500 dilution) and phosphate-buffered serum (PBS:5% milk +0.05% Tween-20) was using for blocking at 25°C room temperature overnight. GAPDH (Sigma, 1:1500 dilution) was used as an internal control. Subsequently, goat anti-rabbit antibody (1:5000 dilution) was using for incubation for 1 hour at 25°C room temperature. Targeted proteins was enhanced with chemiluminescence reagents (Millipore). All experiments were conducted three times independently.