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Supplementary Figure legends
Supplementary Figure 1. PKC does not control the expression of BAFF and its receptor. a, Expression of BAFF-R mRNA in B cells from PKC+/+ or PKC-/- mice. Total RNA was extracted from purified B cells, reverse transcribed using oligo d(T) primers and expression levels of BAFF-R and HPRT were analyzed by semi-quantitative RT-PCR. b, Concentration of BAFF in the serum of the PKC+/+ (open circles), PKC-/- (closed circles) and BAFF-Tg mice (square) was measured by ELISA.
Supplementary Figure 2. PKC does neither control the expression of IL-6 mRNA, secretion of IL-6 nor IL-6-dependent survival of peripheral B cells. a, Expression of IL-6 mRNA in B cells from PKC+/+ or PKC-/- mice. Total RNA was extracted from purified B cells, reverse transcribed using oligo d(T) primers and expression levels of IL-6 and HPRT were analyzed by semi-quantitative RT-PCR. b, Concentration of IL-6 released into the culture supernatant of B cells from PKC+/+ (open circles) and PKC-/- (closed circles) mice in serum-containing medium (left panel) up to 4 days or in the presence of 0.5 g/ml of LPS (right panel) up to 3 days was measured by ELISA. Data represent two independent experiments. c, Purified splenic B cells of PKC+/+ (open symbols) and PKC-/- (closed symbols) mice were incubated in serum-containing medium for 2 days with the indicated amounts of anti-IL-6 (circles) or isotype-matched control (squares) antibodies and the frequency of viable cells was analyzed by staining with Annexin V in combination with 7-AAD. Open circles, PKC+/+; closed circles, PKC-/-. Data are representative for two independent experiments.
Supplementary Information Figure 3. a, PKC does not control the expression of mRNAs encoding anti- or pro-apoptotic proteins in peripheral B cells. Unaltered expression of A1, Bcl-2, and bax mRNA in PKC-/- B cells. Total 2
RNA was extracted from purified wild-type (+/+) or PKC-deficient (-/-) B cells, reverse transcribed using oligo d(T) primers and expression levels of A1, Bcl-2, bax and HPRT were analyzed by semi-quantitative RT-PCR. b, and c, PKC- deficient cells remain sensitive to BAFF. b, Unaltered BAFF-induced NF- B2/p100 processing in PKC-/- B cells. Sorted B cells from PKC+/+ (+/+) and PKC-/- (-/-) mice were pre-incubated in medium for 24 hours before 500 ng/ml of recombinant BAFF was added for either 4 or 24 hours. The presence of p100 and p52 of NF-B2 was determined by Western blot analysis using equal amounts of protein separated by SDS-PAGE, blotted onto nitrocellulose membrane and detected with antibodies against NF-B2 or -actin. Data represent one of two independent experiments. c, Sorted B cells from PKC+/+ (open circles) and PKC-/- (closed circles) mice were pre-incubated in medium for 24 hours before the indicated amounts of recombinant BAFF was added for 3 days and the frequency of viable cells was analyzed by staining with Annexin V in combination with 7-AAD. Data are representative for two independent experiments.