Ecl kit

For the validation of the differentially expressed proteins, western blotting was used for quantitative analysis. Cartilage tissues were lysed by 2% sodium dodecyl sulfate with 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride, then the lysates were centrifuged. The supernatants were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% skimmed milk, the membrane was then analyzed using specific antibodies and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences, Little Chalfont, UK). The primary antibodies to Indian hedgehog (IHH, ab52919), type I collagen (COL1A1, ab138492), type II collagen (COL2A1, ab188570), aggrecan (ACAN, ab3778) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab9894) were obtained from Abcam (Cambridge, UK). Protein signals were detected using secondary HRP-conjugated anti-mouse or anti-rabbit antibodies by the enhanced chemiluminescence (ECL Kit, Amersham Biosciences, Little Chalfont, UK). The full-length images of individual blots were displayed in Fig. S1.

We performed immunoprecipitation as described previously [72 (link)]. In brief, total cell lysates were collected and then were immunoprecipitated by incubation with antibodies against Flag (1:100, Cell Signaling Technology, Boston, MA, USA) and HA (1:100, Cell Signaling Technology). SDS–PAGE was used to separate immunoprecipitates and then was blotted onto a polyvinylidene difluoride (BioRad Laboratories, Hercules, CA, USA) membrane. The membrane was incubated with antibodies against HA (1:2000, Cell Signaling Technology) and Flag (1:2000, Cell Signaling Technology) respectively and visualized by enhanced chemiluminescence (ECL Kit; Amersham Biosciences, Slough, Buckinghamshire, UK). Whole-cell lysates were separated by SDS–PAGE and blotted on polyvinylidene difluoride membranes (Millipore) for western blotting analysis reprobed with appropriate peroxidase-conjugated HA and Flag antibodies. Blots were visualized by enhanced chemiluminescence (ECL Kit; Amersham Biosciences).

180×10⁴ HUVECs were electroporated and seeded on a six-well plate. After 24 h, the medium was changed and the cells incubated for 48 h. The cells were lysed in 100 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 1 mM DTT, 1% Triton X-100) with protease inhibitor cocktail (NACALAI) for 30 min on ice, centrifuged at 14,500 rpm for 10 min at 4°C, and the supernatants recovered. The protein concentration was measured using Bradford Protein Assay kits (Bio-Rad), and 30 µg of each protein was electrophoresed using 10% SDS-PAGE. The proteins were transferred to a PVDF membrane (Immobilon-P, Merck), blocked with 5% skim milk, and incubated with anti-SMAP1 and AGFG2 antibodies. The peroxidase-conjugated secondary antibody was incubated and reacted using ECL kits (Cytiva).The signal was detected using ChemiDoc XRS+ (Bio-Rad). The membrane was stripped using stripping buffer (strong, NAKALAI) for detecting the β-actin signal as a loading control.
To investigate vWF processing, 50×10⁴ HUVECs were electroporated and seeded on a six-well plate. After 24 h, the medium was changed and the cells incubated for 48 h. The cells were scraped in 100 µl of lysis buffer, lysed by being drawn into and expelled from a 23 G needle five times, and kept on ice for 30 min. After centrifugation, the supernatant was taken and subjected to western blotting.