P pkc

The C2C12 myoblasts were treated with different concentrations (25 µg/mL and 12.5 µg/mL) of IC or AC for 30 min before stimulating them with 100 ng/mL of LPS for 24 h. The whole-cell lysates were prepared using NP-40 lysis buffer (1 µg/mL of leupeptin, 1 mM of PMSF, 2.5 mM of sodium pyrophosphate, 1 mM of Na₃VO₄, 1 mM of β-glycerol phosphate, 1% sodium deoxycholate, 1% NP-40, 1 mM of EGTA/EDTA, 20 mM of Tris-HCl (pH 7.4), and 150 mM of NaCl) and the protein concentrations in each sample were determined using a Bradford assay. The antibodies against N-cadherin (Abcam Cat# 2447-1, RRID:AB_1267002), p-PKC (Santa Cruz Biotechnology Cat# sc-136018, RRID:AB_2168554), actin (BD Biosciences Cat# 612656, RRID:AB_2289199), and β-catenin (Cell Signaling Technology, Danvers, MA, USA. Cat# 19807, RRID:AB_2650576) were used at 1:1000 dilutions. The procedure followed for Western blot has been described in detail in our previous paper by Chao et al. [30 (link)].

Antibodies against the following proteins were used: PLD2 (sc-515744), BAX (sc-7480), XIAP (sc-55550), Bim (sc-374358), VEGF (sc-7269), EGFR (sc-373746), p-EGFR (sc-81488), AKT (sc-81434), p-AKT (sc-377556), PKCζ (sc-17781), pPKC (sc-12894R), PKCδ (sc-8402), p-PKCδ (sc-377560) and α-tubulin (sc-8035, Santa Cruz Biotechnology, Dallas, TX, USA), acetyl-Histone 4 (06-866, EMD Millipore, Burlington, MA, USA), ERK (#9102), p-ERK (#9101S), JNK (#9252), p-JNK (#4668), IκBα (#4814), p-IκBα (#2859), Src (#2109), p-Src (#2101), S6K (#2708), p-S6K (#9234), p38 (#9212), p-p38 (#9211) and cleavaged caspase 3 (#9661, Cell Signaling, Danver, MA, USA). The signal densities on the blots were measured with Image J (Wayne Rasband) and normalized using anti-α-tubulin antibody.

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.