Figure 3
Anti-C2Cat antibodies immunoprecipitate more cPKC upon activation with PMA.
(A) HEK293T cells were transfected with either WTPKCβΙ or ΔNPSPKCβΙ and treated with 100 nM PMA for 15 minutes. Fixed cells were then incubated with anti-PKCβΙ V5 domain antibodies and subsequently with anti-rabbit antibodies labeled with Alexa 555. (B) To evaluate the amount of active (membrane bound) PKCβΙ in HEK293T transfected cells. Cells treated or non-treated with 100 nM PMA were fractionated and probed for PKCβΙ in the particulate fraction by Western blot with anti-PKCβΙ-V5. (C) Transfected HEK293T cells were treated with 100 nM PMA and control cells were immunoprecipated with anti-C2Cat antibodies and probed for PKCβΙ with anti-PKCβΙ-V5 (upper panel). Total lysates were probed with anti-PKCβΙ to evaluate transfection levels and for α-tubulin to evaluate the total amount of protein loaded. A representative blot of n = 3 is shown for (B,C). Quantitative analysis of the average of three independent experiments is shown normalized to non-treated cells, which was set to 1. Statistical significance was determine by ANOVA-Bonferroni test where **p < 0.01 and *p < 0.05.
