Phospho ser pkc substrate antibody

STC-1 cells (2 × 10⁶) were lysed in 100 μl of RIPA buffer containing a protease and phosphatase inhibitor cocktail (Roche). Cell lysates were quantified, and an aliquot was resolved using 10% SDS-PAGE gels. Western blot analyses were performed using antibodies raised against the respective total and phospho-proteins, namely, for Protein kinase C, calcium calmodulin kinase ii (CaMKii) (PKC) (total anti-CaMKIIα, 6G9, catalog-# MA1–048, Thermo Fisher; phospho anti-pT286-CaMKII catalog-# sc-12886-R, Santa Cruz Biotechnology), for extracellular-regulated kinase (ERK)(total catalog-# 4695, phospho catalog-# 4376, both from Cell Signaling Technology), and a phospho-(Ser) PKC substrate antibody (catalog-# 2261 from Cell Signaling Technology). Protein expression was normalized by β-actin (catalog-# 3700, Cell Signaling Technology) or GAPDH (catalog-# ab8245, Abcam). Enhanced chemiluminescent reagents (GE Healthcare) were used to detect antigens after electrophoretic transfer of proteins resolved by SDS-PAGE to PVDF or nitrocellulose membranes, respectively.

To verify the expression of the DGK isotypes and detect PKC substrate, 50 μg of proteins from cell homogenates were separated by 8.75% SDS-PAGE gel under reducing conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane, Immobilon (Darmstadt, Germany). The membrane was blocked by incubation in Tris-buffered saline with detergent Tween 20 (TBST) containing 10% (w/v) dried milk for 1 h at 25°C. The TBST contained the following: 50 mM Tris, 150 mM NaCl, and 0.05% (w:v) Tween 20, pH 7.4. For detection of DGKs, the membrane was incubated with monoclonal anti-FLAG antibody (1:1000) in TBST, at 25°C for 1 h with gentle agitation. For the detection of PKC substrates, the PVDF membrane was incubated with Phospho-(Ser)-PKC substrate antibody, Cell Signaling (Danvers, MA), (1:1000) in TBST overnight at 4°C with gentle agitation. The membranes were then incubated with alkaline phosphatase-conjugated mouse or rabbit secondary antibody (1:10,000) diluted in TBST with 2% (w/v) dried milk, for 1 h at 25°C. After three 15 min washes with TBST, the membrane was briefly incubated in chemiluminescent alkaline phosphatase substrate, Applied Biosystems (Poster City, CA). The immunoreactivity was detected using a Fuji LAS 4000.