Chemiluminescence hrp substrate kit

Three to four million cells were lysed in modified radio immunoprecipitation assay (RIPA) buffer (25 mM Tris HCl pH 7.6, 151.5 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1x Complete mini protease inhibitor (Roche, Indianapolis, IN), 1 mM PMSF) for 40 minutes on ice. The lysates were clarified by centrifugation (15 minutes at 14000 rpm). 30–40 μg of lysate was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore, Billerica, MA). Membranes were blocked with either 5% skim milk diluted in PBS-Tween (0.1%) or Odyssey blocking buffer (Licor Biosciences, Lincoln, NE) then incubated with anti-EGFR, anti-β-actin or anti-α-tubulin diluted in blocking buffer at 4°C overnight. The filters were incubated with secondary antibodies conjugated to horse radish peroxidase and the immunocomplexes detected with the Chemiluminescence HRP Substrate kit (Millipore) or incubated with IRDye conjugated secondary antibodies and the immunocomplexes detected by fluorescence using the Licor Odyssey Classic (Licor Biosciences).

The proteins of exosomes were extracted with RIPA buffer (Beyotime, China) and centrifuged at 11,809 g for 15 min at 4°C. A small amount of supernatant was quantified using the bicinchoninic acid assay (BCA) kit (Beyotime), and the remaining proteins were separated by SDS‐PAGE and transferred onto a PVDF membrane (Millipore) according to standard methods. After blocking with 5% non‐fat milk for 2 h, the membrane was incubated overnight at 4°C with primary antibodies for CD63 (proteintech). After washing the non‐bound primary antibodies, the membrane was incubated with secondary HRP‐conjugated anti‐mouse or anti‐rabbit antibodies (Beyotime). The antigen‐antibody reaction was detected by a chemiluminescence HRP substrate kit (Millipore).

Whole-cell lysate was extracted from VSMCs or neutrophils using RIPA Lysis Buffer (C1053, Applygen, Beijing, China). The protein concentration of the samples was measured by Pierce™ BCA Protein Assay Kit (23209, Thermo Fisher Scientific, Waltham, MA, USA). An equal amount of protein per sample and molecular weight markers was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were blocked and probed for primary antibodies against citH3 (1:1000 dilution; ab5103, Abcam, USA), GAPDH (1:2000 dilution; 10494-1-AP, Proteintech, Chicago, IL, USA), p38 (1:1000 dilution; 8690s, CST, Danvers, MA, USA), p-p38 (Thr180/Tyr182) (1:1000 dilution; 4511s, CST, USA), JNK (1:1000 dilution; 9252s, CST, USA), and p-JNK (Thr183/Tyr185) (1:1000 dilution; 4668s, CST, USA) overnight at 4°C. Anti-mouse (1:2000, 7076S; Cell Signaling, MA, USA) and anti-rabbit secondary antibody (1:2000, 7074S; Cell Signaling, MA, USA) conjugated with horseradish peroxidase for 1 h at room temperature. Chemiluminescence HRP Substrate Kit (Millipore, Bedford, MA, USA) was used to perform immunodetection analysis. The mean band intensity was measured with ImageJ.

Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich, USA) and quantified with Micro BCA Protein Assay Kit (Thermo-Fisher, USA). Ten micrograms of protein were electrophoresed by 15% SDS polyacrylamide gels, transferred to PVDF membranes (Millipore, GER), and then blocked in 5% skim milk for 1 h at room temperature, incubated with primary antibodies at 4 °C overnight. Secondary antibodies were labeled with horseradish peroxidase and detected by chemi-luminescence HRP substrate kit (Millipore, GER). The antibodies used for immunoblotting were ID3 (sc-374287; Santa Cruz, USA), E47 (sc-416; Santa Cruz, USA), and β-actin (A1978; Sigma-Aldrich, USA).