A2856

Western blot analysis was performed as previously described [6 ]. Regular Western blot analysis was performed using LV lysates that were prepared using RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protein inhibitor mixture (Sigma-Aldrich)). In addition, Western blot analysis of protein aggregation was performed using LV detergent-soluble and -insoluble lysates. Briefly, detergent-soluble fractions of LV tissues were obtained using a 2% Triton X-100 lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 2% Triton X-100, and a protein inhibitor mixture (Sigma-Aldrich)). The insoluble fractions were further solubilized using the 2% Triton X-100 lysis buffer supplemented with 1% SDS. Primary antibodies included anti-LC3 (L7543, Sigma-Aldrich), anti-P62 (ab91526, abcam), anti-NQO1 (sc-376023, Santa Cruz Biotechnology, Inc.), Anti-ATG5 (A2859, Sigma-Aldrich), anti-ATG7 (A2856, Sigma-Aldrich), anti-LAMP1 (ab24170, Abcam), anticathepsin D (2284, CST), and anti-Ub (sc-8017, Santa Cruz Biotechnology, Inc.). Ubiquitinated proteins with molecular weights from the top to 35 kDa on the membranes of immunoblots were quantified

Femoral arteries and hearts were fixed in 4% formalin for 24 h and paraffin embedded. Transversal sections of the heart were stained with masson’s trichrome staining to determine cardiac fibrosis. Atg7 on femoral artery segments was assessed by immunohistochemistry using anti-Atg7 (Sigma-Aldrich, A2856) monoclonal antibody. Transversal sections of femoral artery segments were stained with hematoxylin-eosin (H&E) to calculate media thickness. Images were acquired with Universal Graph 6.1 software using an Olympus BX40 microscope and quantified with Image J software.

l‐arginine (A8094), the primary antibodies against Rep2 (029628), LC3 (L7543) and ATG7 (A2856), as well as horseradish peroxidase‐conjugated secondary antibodies (goat anti‐rabbit, (A0545); goat anti‐mouse, (A9044)) were purchased from Sigma. The primary antibodies against GAPDH (sc‐32233), NQO1 (sc‐32793), NRF2 (sc‐13032), RAB7 (sc‐376362) and phospho‐S6 (sc‐293144) were purchased from Santa Cruz. LAMP1 (9091), mTOR (2983), phospho‐mTOR (5536), S6K (9202), phospho‐S6K (9234), and S6 (2217) antibodies were all obtained from Cell Signaling. The primary antibody against SQSTM1/p62 (89‐015‐843) was obtained from Abnova, and the SNAP29 antibody from Proteintech (12704‐1‐AP). CHML Flexitube siRNA constructs were purchased from Qiagen. The A549, H1299, A375 and MDA‐231 cell lines were all purchased from the American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium (DMEM; MT10014CV) was purchased from Corning. DMEM low glucose without amino acids was purchased from US Biological Life Sciences (D9800‐13). (FBS; S11150H) was purchased from Atlanta Biologicals. l‐glutamine (25030081) and penicillin‐streptomycin (15140722) were obtained from Gibco.