Atg10

Immunoprecipitation and Western blot assays were performed according to the previously described procedures [4 (link)]. Antibodies Runx2 (Abcam, UK, 1:1000), ALP (Abcam, UK, 1:2000), SM22ɑ (Abcam, UK, 1:1000), p16 (Proteintech, USA, 1:500), p21 (Cell signaling, USA, 1:1000), LC3-II (Abcam, UK, 1:500), SQSTM1 (Abcam, UK, 1:1000), Atg10 (Abcam, UK, 1:1000), Bhlhe40 (Proteintech, USA, 1:500), GAPDH (Abcam, UK, 1:4000), and β-actin (Proteintech, USA, 1:2000) were used in this study. HA-VSMCs were transfected as indicated and lysed in an immunoprecipitation buffer. The lysates were centrifuged for 20 min. Supernatants were incubated with specific antibodies overnight at 4℃ and protein A/G Agarose beads for 4 h. The immunoprecipitates were washed three times with PBS, and then analyzed via SDS-PAGE. The protein bands were visualized using ECL-Plus Western blot detection kit (Amersham BioSciences, UK).

The total protein from cells was extracted with RIPA lysate. The homogenates, which contained 20 µg of protein, were run on 12–15% SDS-PAGE gels and then transferred to a PVDF membrane (Millipore, Shanghai, China). The PVDF membrane was blocked with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies, including LC3 (1:300; Abcam), Atg10 (1:1000; GTX), and GAPDH (1:3000; Abcam).

After incubation cells were lysed directly into sample buffer (50 mm Tris-HCl buffer pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue) containing 100 mm maleimide to preserve protein oxidation. Protein samples were resolved on SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes by western blotting (TransBlot Turbo, Bio-Rad). Membranes were blocked in milk dissolved in phosphate-buffered saline (PBS) supplemented with 1% Tween before incubation with primary antibodies. Membranes were developed using enhanced chemiluminescence substrate (ThermoFisher) and detected using photographic film, which was developed using an automatic processor (Fuji RG II). Samples were immunoblotted for LC3 (1:1000, Cell Signaling), Atg3 (1:5000, Abcam), Atg7 (1:1000, Cell Signaling), Actin (1:10,000, Sigma), Atg5 (1:1000, Cell Signaling), Atg12 (1:1000, Cell Signaling), Atg4B (1:1000, Cell Signaling), Atg10 (1:1000, Abcam), GABARAPL1 (1:1000, Cell Signaling), GABARAPL2 (1:1000, Cell Signaling), cGMP-dependent protein kinase (PKG, 1:1000, ENZO Life Sciences), Myc-tag (1:1000, Cell Signaling), p62 (1:500, Cell Signaling), caspase-3 (1:1000, Abcam), peroxiredoxin-SO3 (1:500, AbFrontier) and GFP (1:2000, Miltenyi Biotec). All uncropped western blots can be found in Supplementary Fig. 6.