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  Indian J Med Microbiol
 

Figure 2: Western blot showing detection of PKC isoforms βI and βII in DRG neurons by their respective antibodies. Arrowheads indicate expected kD of each individual PKC isoform. Method: DRG were isolated in cold PBS and centrifuged at 700×g for 5 min at 4°C. The supernatant was aspirated and DRG pellet re-suspended in RIPA Buffer [10 mM Tris-HCl (pH 7.4), 1% NP40, 0.1% sodium deoxycholate (DOC), 0.1% SDS, 150 mM NaCl, 1 mM EDTA] with an added protease inhibitor cocktail and homogenized completely at 1,600 rpm in a homogenizer and kept on ice for 1 h with intermittent vortex mixing. Homogenized and lysed DRG were centrifuged at 10,000 ×g for 1 h at 4°C. Aliquot of supernatant was analyzed for total protein content using Protein Assay. The remainder of the supernatant of the cell lysate was then heated for 5 min at 95°C in an SDS sample buffer and protein samples thus obtained were separated by SDS-PAGE (7.5% polyacrylamide). Precision Plus All Blue protein standard (Bio-Rad) was used as protein marker. Membranes (PVDF, Millipore) were probed with primary antibodies rabbit anti-PKCβI and rabbit anti-PKCβII at 1:100 dilutions. Secondary antibody consisted of anti-rabbit HRP (1/5000) (Promega). Proteins were detected using the ECL system (Bio-Rad).

Figure 2: Western blot showing detection of PKC isoforms βI and βII in DRG neurons by their respective antibodies. Arrowheads indicate expected kD of each individual PKC isoform. Method: DRG were isolated in cold PBS and centrifuged at 700×g for 5 min at 4°C. The supernatant was aspirated and DRG pellet re-suspended in RIPA Buffer [10 mM Tris-HCl (pH 7.4), 1% NP40, 0.1% sodium deoxycholate (DOC), 0.1% SDS, 150 mM NaCl, 1 mM EDTA] with an added protease inhibitor cocktail and homogenized completely at 1,600 rpm in a homogenizer and kept on ice for 1 h with intermittent vortex mixing. Homogenized and lysed DRG were centrifuged at 10,000 ×g for 1 h at 4°C. Aliquot of supernatant was analyzed for total protein content using Protein Assay. The remainder of the supernatant of the cell lysate was then heated for 5 min at 95°C in an SDS sample buffer and protein samples thus obtained were separated by SDS-PAGE (7.5% polyacrylamide). Precision Plus All Blue protein standard (Bio-Rad) was used as protein marker. Membranes (PVDF, Millipore) were probed with primary antibodies rabbit anti-PKCβI and rabbit anti-PKCβII at 1:100 dilutions. Secondary antibody consisted of anti-rabbit HRP (1/5000) (Promega). Proteins were detected using the ECL system (Bio-Rad).