Ecl detection kit

N2a cells and the ipsilateral cortex of SD rats were processed for Western blot as described (Fan et al., 2016 (link)). Immunoblot analyses were performed using the following primary antibodies: anti-SPCA1 (ab126171, Abcam) and anti-β-actin (60008-1-Ig, Proteintech, United States). The anti-rabbit IgG and anti-mouse IgG secondary antibodies were obtained from Proteintech. The proteins were visualized using an enhanced chemiluminescent (ECL) detection kit (Advansta Inc., United States).

Co-immunoprecipitation experiments were performed with lysates from cultured BMDMs following the immunoprecipitation protocol provided by the manufacturer Abcam Inc. In brief, cell lysates were harvested on ice in non-denaturing lysis buffer (20 mM Tris HCl pH8, 137 mM NaCl, 1% Triton X-100, and 2 mM EDTA in DI water) containing PIC (Sigma #P8340). Lysates were incubated under gentle agitation for 30 min. at 4°C following by centrifugation. Protein concentrations were determined using the BioRad DC Protein Assay kit (#5000112) and a BSA standard. 50 μg of lysate from each treatment was added to a tube containing Protein G Agarose beads (Active Motif #37449) linked to antibody, and incubated under gentle agitation overnight. 50 μL of Protein G Agarose bead slurry was incubated with 5 μg of antibody under gentle agitation at 4°C for 4 h and washed in lysis buffer prior to addition of lysate. Antibodies against Nrf2 (ThermoFisher #PA5-27882) and Keap1 (ProteinTech #10503-2-AP) were used. Beads were washed the following day and complex eluted in SDS Buffer and boiled before Western blot. Blots were probed with the opposite antibody (Nrf2 on Keap1 IP blot and Keap1 on Nrf2 IP blot). Blots were detected using a conformation-specific anti-rabbit HRP-linked IgG secondary antibody (Cell Signaling #5127) and Advansta ECL detection kit.

HCC cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail (Bimake, Shanghai, China). The protein concentrations were quantified with the BCA Protein Assay Kit (Beyotime). Protein samples were separated by 10% SDS-PAGE and then electroblotted onto PVDF membranes (Millipore, VT, USA). After blocking with 5% skim milk in TBST for 2 h, the membranes were then incubated with the primary antibody overnight at 4°C. Corresponding HRP-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) were used to incubate the membranes for 1 h at room temperature. The protein bands were visualized using the ECL Detection Kit (Advansta, CA, USA). The primary antibodies used in this experiment included anti-Wnt1 antibody (Proteintech Europe, Manchester, UK), anti-β-catenin antibody (Abcam, MA, USA), anti-c-Myc antibody (Abways Technology, Shanghai, China), anti-E-cadherin antibody and anti-N-cadherin antibody (Cell Signaling Technology, Danvers, USA), anti-Vimentin antibody (Bimake, Houston, USA), anti-Snail antibody (Wanleibio, Shenyang, China), and anti-GAPDH antibody (Abways Technology, Shanghai, China).

EVs, cells and matrigel tissue were processed for Western blot as described [12 (link), 13 (link)]. Immunoblot analyses were performed using the following primary antibodies against Calnexin (1:1000, ProteinTech, China), TSG101 (1:1000, ProteinTech, China), CD81 (1:1000, ProteinTech, China), HIF-1α (5 µg/mL, abcam, UK), VEGF (5 µg/mL, abcam, UK), TP53(1:1200, ProteinTech, China), GAPDH (1:3000, ProteinTech, China).The anti-rabbit IgG and anti-mouse IgG secondary antibodies were obtained from Proteintech. The proteins were visualized using an enhanced chemiluminescent (ECL) detection kit (Advansta Inc., United States).

The cells were lysed using a RIPA buffer (iNtRON) containing protease and phosphatase inhibitor cocktails. The cell lysates were resolved using 10% SDS-PAGE and transferred to nitrocellulose membranes, which were blocked for 1 h at room temperature (approximately 23 °C) using phosphate-buffered saline with 0.01% Tween-20 (PBST, Merck Korea, Seoul, Korea) containing 10% fat-free dried milk. The blocked membranes were then incubated overnight with primary antibodies at 4 °C, followed by further incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were developed using the enhanced chemiluminescence (ECL) detection kit (Advansta, San Jose, CA, USA) and visualized using a luminescent image analyzer (Amersham imager 680, Cytiva Korea, Incheon, Korea).

The TNC samples were homogenized in RIPA lysis buffer for 1 h. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). The proteins were separated on an SDS-PAGE gel, electrophoresed, and transferred to PDVF membranes. Following transfer, the membranes were blocked for 2 h at room temperature in TBST containing 5% non-fat milk and then incubated overnight at 4 °C with the appropriate antibodies diluted in TBST: rabbit anti-P2X4R (1/500, Abcam), rabbit anti-EKR1/2 (1:1000, CST), mouse anti-BDNF (1:1000, Abcam) and rabbit anti-p38-MAPK and p-p38-MAPK (1:1000, CST). The next day, after three washes in TBST, the membranes were incubated with specific HRP-conjugated secondary antibodies for 2 h at room temperature and revealed with an ECL detection kit (Advansta). β-Actin (1:9000, Proteintech, China) was used as a control to normalize protein levels.