Protein lysis buffer

Six-well dishes (60 mm diameter) of cells were treated as described in the figure legends, followed by lysis in protein lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Cell lysate was resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. Samples were normalized to β–actin (1:10,000 dilution, A5316, Sigma-Aldrich, St. Louis, MO, USA) expression and probed with antibodies for VN (1:1000 dilution, GTX103475, GeneTex, Irvine, CA, USA) and CLU (1:1000, A241, Quidel, San Diego, CA, USA). Blots were visualized by horseradish peroxidase-conjugated antibodies (Sigma-Aldrich) and chemiluminescence (Thermo Fisher Scientific).

In this study, two measurements were performed to detect the ROS production in the RVLM tissue. After RVLM tissue was punched and weighed from the rat which was euthanized (pentobarbital sodium, 300 mg/kg, i.p.), 80 μL Protein Lysis Buffer (Cell Signaling Technology, USA) was added into the test tube and tissue was polished by electric homogenizer and then centrifuging for 20 min. Supernatant was collected for analysis by lucigenin chemiluminescence quantitative kit (Genmed Scientifics Inc., USA, GMS10113.5) and dihydroethidium (DHE). We can complete lucigenin chemiluminescence quantitative detecting according to the instructions. DHE, ROS sensitive fluorescent dye, brain tissues (15 μm thick) were incubated at 37°C with DHE (5 μmol/L) for 30 min. Sections were washed in 0.1 M PBS (3 × 1 min) and then examined by confocal laser scanning microscope (Fuji Film, Japan) and the image was captured at red fluorescence microscope around the RVLM and was evaluated using LAS-AF-Lite software [24 (link)].

The proteins were extracted from the cells in a protein lysis buffer (Cell Signaling Technology, MA, USA) and subsequently centrifuged at 22,000 g for 5 min at 4°C to remove cellular debris. After boiling for 5 min, the lysate samples were separated by a 12% SDS-PAGE electrophoresis and electrotransferred to a PVDF membrane. The membrane was blocked in TBST containing 5% BSA and incubated with anti-P2X7, P2Y1, P2Y2, P2Y11, VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MYH-11 (1 : 500), or GAPDH antibodies (1 : 5,000) (Santa Cruz Biotechnology, CA, USA) overnight at 4°C. The membranes were washed three times with TBST and incubated with the secondary antibodies (1 : 5,000) (CALBIOCHEM, CA, USA) for 60 min at RT. After washing with TBST, immune-detection was accomplished by using the Luminata Forte Western HRP substrate (Merck Millipore, MA, USA) and images were taken using Bio-Rad Chemidoc system.

Murine epidermis was dissociated from the dermis before isolation of bulk RNA and protein. Time points were selected as to not include the anagen phase of the hair cycle, which compromises the dissociation process. Following euthanasia of 21-day-old mice, the skin was dissected, and the underlying fat pad was removed using a scalpel. The resulting tissue was floated dermis side down in dispase (5 U/ml; Corning) in PBS for 40 min at 37°C. The epidermis was then removed using a scalpel, and for RNA, the epidermis was flash-frozen in TRIzol and stored at −80°C until RNA isolation. RNA was extracted using an RNeasy kit (#74104, QIAGEN) at the same time and date for all mice belonging to a single experimental cohort (i.e., RNA-seq) regardless of the date of murine euthanasia to reduce batch effects and stored at −80°C. For protein, epidermis was placed directly into cold PBS and centrifuged at 4°C for 5 min at 2500 rpm. To the resulting pellet, protein lysis buffer (Cell Signaling Technology) containing a protease inhibitor cocktail was added, and the mixture was homogenized, sonicated, rotated at 4°C for 10 min, and then centrifuged at 4°C at full speed for 10 min. Lysates were quantified using the Bradford assay. Frozen lysates were stored at −80°C.

Immunoblot analysis was performed as we have described previously [8 (link),29 (link),31 (link)]. Cells were collected and lysed in protein lysis buffer (Cell Signaling, Danvers, MA, USA) with protein and phosphatase inhibitors (Thermo-Fisher/Pierce, Rockford, IL, USA) and cleared by centrifugation. Protein concentration of the supernatant was quantified, boiled in laemmli sample buffer for 5 min. Cell lysate (20ug) was separated on gradient or 10% PAGE gels, transferred to nitrocellulose, and blocked with 5% BSA before overnight incubation with primary antibodies, diluted 1/1000 at 4 degrees C, followed by HRP conjugated secondary antibody incubation, diluted 1/2000, for 1 h at room temperature (Supplementary Table S1). An ECL chemiluminescence kit (GE Healthcare/Sigma, St. Louis, MO, USA) was used for visualization on X-ray film, and bands were quantified (as integrated density) using ImageJ software (NIH, Bethesda, MD, USA).

Protein from kidney tissues was extracted by using the protein lysis buffer(Cell Signaling,Danvers, MA, USA) and then quantified by the Bradford assay (Bio-Rad, USA) . An equal amount of protein was separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Biosciences, USA). After blocking non-specific binding with 5% skim milk in TBST for 1 h at room temperature, membranes were then incubated overnight at 4 °C with the primary antibody against Flna (Cell Signaling, Danvers, MA, USA), Col. III and Col. I (Southernbiotech, USA), Trem1 and Pvalb (boster,Wuhan, China), β-actin(Santa Cruz, USA), followed by horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The ratio for the protein examined was normalized against β-actin and was expressed as the mean ± SD.