β actin

Freshly excised cancer specimens were taken for Western blot analysis. BGC-803 cells (10⁵ cells/well) were plated in 24-well plates and cultured for 24 h. After removing the medium, RPMI-1640 containing 0.1, 0.2, and 0.3 mM BAPN, or 2.5, 5, and 10 nM LOX was added and incubated for 24 h.
According to the manufacturer's instructions, total protein extract from the specimens and cultured cells was obtained using the total protein extraction kit (Key GEN Bio TECH, Jiangsu Province, China). After 10% polyacrylamide gel electrophoresis, isolated proteins were transferred to PVDF membranes (EMD Millipore, Billerica, MA) and further blocked in a 5% skim milk solution at room temperature for two hours. They were incubated independently with 1:100 rabbit anti-LOX, 1:200 rabbit anti-PDGFR, 1:50 rabbit anti-PDGFRα, or 1:50 goat anti-PDGFRβ antibody (Santa Cruz Biotechnology), or 1:1000 rabbit anti-actin (Key GEN Bio TECH, Jiangsu Province, China) at room temperature for 3 h and then treated with HRP-labeled anti-rabbit or anti-goat IgG. After DAB staining, the gray value of the target protein bands was quantified by Quantity One software, and the relative protein expression was calculated as follows: protein relative expression = target protein expression intensity/β-actin protein expression intensity.

The renal cortex tissues from the diabetic models or podocytes were lysed on ice for 30s with a RIPA lysis buffer. Equal protein concentrations were loaded using sodium dodecyl sulphate (SDS) -polyacrylamide gel electrophoresis and transferred to an immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking by 5% BSA solution, the membranes were then incubated overnight at 4 °C with primary antibodies against nephrin and fibronectin (1:500 dilution, Santa Cruz, USA), collagen I (1:1000 dilution, Abcam, USA), α-SMA (1:1000 dilution, Abcam, UK), GPR43 (1:1000 dilution, Merck, Germany), AMPKα (1:1000 dilution, Cell Signaling, USA), pAMPKα (Thr172) (1:1000 dilution, Cell Signaling, USA), Akt (1:1000 dilution, Cell Signaling, USA), pAkt (Ser473) (1:1000 dilution, Cell Signaling, USA), PKC (1:500 dilution, Santa Cruz, USA), pPKC (1:500 dilution, Santa Cruz, USA), PLCγ1 (1:1000 dilution, Abcam, UK), pPLCγ1 (1:1000 dilution, Abcam, UK) and β-actin (1:3000 dilution, KeyGEN, Netherlands) followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG for 1h. Quantification was performed by measuring the intensity with the ImageJ software.

FeCl₂·4H₂O, FeCl₃·6H₂O, diethyleneglycol (DEG), Zn(NO₃)₂·6H₂O, and NaOH were purchased from Shanghai Chemical Reagents Co (Shanghai, China). Dox was acquired from Hisun Phamaceuticals, Zhejiang, China. Cell Counting Kit-8 (CCK-8) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma (St Louis, MO). RPMI-1640 culture medium, fetal bovine serum (FBS), and cultured plate were purchased from Gibco (Gibco/BRL, Carlsbad, CA). The antibodies of Bax, Bcl-2, Casepase 3, β-actin and horseradish peroxidase-conjugated IgG antibody were obtained from Nanjing KeyGen Biotech. Inc (Nanjing, China). All other reagents were of analytical grades.