Figure 1

Specificity of the anti-PKC isozyme monoclonal antibodies. Lysates from PBMC from human blood of adult volunteers were subjected to Western blot analysis using isoenzyme-specific monoclonal anti-PKC antibodies as indicated in figures. These were developed with an HRP-conjugated anti-mouse/anti-rabbit Ig antibody. (a) Shows the reactivity of antibodies recommended for flow cytometry. The blots show either one or two samples (lanes 1 and 2) with blots stripped and reprobed to detect GAPDH. These antibodies either did not show any immunoreactivity towards the intended PKC isozyme or they showed non-specific binding to multiple proteins. Antibodies: anti-PKCε, -PKCβI, -PKCλ/ι and -PKCδ antibodies were from clones E-5, E-3, E-7 and G-9; (Supplementary Table S1). (b) Specificity of antibodies selected for flow cytometry assay. These anti-PKC antibodies were selected based on their specificities, as they each showed specific binding to a single protein band. The clones for the anti-PKCα (H-7), βI (EPR18512), βII (F-7), δ (EPR17075), ε (EPR1482(2)), η (EPR18513), θ (E-7), ζ (H-1), ι/λ (H-12) and μ (EP1493Y) are shown in parentheses (see also Supplementary Table S1). The blots are presented individually for each staining, intact without splicing, with lanes in their entirety.