Anti pkc

After 72 h of incubation with or without canagliflozin in the presence of 5.5 mM or 25 mM glucose, MCs were collected and sonicated in 400 µl of solubilizing solution containing 9 M urea, 2% Triton X-100, and 1% dithiothreitol. Then, 100 µl of 10% lithium dodecyl sulfate was added to the solubilized membrane fraction and sonicated again. Subsequently, proteins were separated discontinuously on 7.5% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking nonspecific binding, the membranes were incubated overnight at 4 °C with rabbit anti-NOX4 (1:1000; Abcam), anti-PKC (1:250; Abcam), anti-pan phospho-PKC (1:1000; Cell Signaling Technology), anti-SGLT2 (1:500; Abcam), anti-TGF-β (1:500; Cell Signaling Technology), or anti-fibronectin (1:5000; Abcam) antibodies, followed by HRP-conjugated donkey anti-rabbit IgG antibody (1:10,000; Amersham) as a secondary antibody. The membranes were also incubated with goat anti-β-actin HRP-conjugated antibody (1:5000; Santa Cruz) to ensure equal protein loading of the lanes. We used the ECL Plus system (Amersham) to detect antibody binding.

Fresh tissue samples were collected from the retina. To analyze the expression levels of respective proteins, equal amounts of total proteins were subjected to 10% (w/v) SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Membranes were then blocked with 5% skim milk for 2 hours at room temperature and probed with anti-VEGF (1 : 500; Santa Cruz Biotechnology, Dallas, TX), anti-Flk-1 (1 : 1000; Santa Cruz Biotechnology, Dallas, TX), anti-CREB (1 : 1000; Millipore, Darmstadt, Germany), anti-PKC (1 : 750; Abcam, Cambridge, UK), anti-β actin (1 : 1000; Santa Cruz Biotechnology, Dallas, TX), anti-GAPDH (Abcam, Cambridge, UK), anti-nNOS (1 : 1000, BD Biosciences, USA), anti-iNOS (1 : 750, Santa Cruz Biotechnology, Dallas, TX), and anti-eNOS (1 : 750, Santa Cruz Biotechnology, Dallas, TX) antibodies at 4°C overnight. Subsequently, membranes were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA) or rabbit anti-rat (Thermo Fisher Scientific, Waltham, MA) IgG for 2 hours at room temperature after three times TBST washing and then reacted with a prolight HRP agent (Santa Cruz Biotechnology, Dallas, TX). The result of chemiluminescence was recorded with an imaging system and semiquantified using the ImageJ software (National Institutes of Health, Bethesda, MD).