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1.
Figure 8.

Figure 8. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

(A) Unaltered BAFF-induced NF-B2/p100 processing in the absence of PKC. The presence of p100 and p52 was determined by Western blot analysis using an antibody against NF-κB2. Protein loading was controlled by Erk (p44/p42) expression. (B) Unaltered BAFF-induced cytoplasmic retention of PKCδ in the absence of PKCβ. Cytoplasmic and nuclear extracts were prepared from PKCβ+/+ and PKCβ-deficient B cells at time 0 and after 24 h of incubation in the absence (−) or presence (+) of BAFF. PKCδ expression was assessed by Western blotting. Fraction purity and protein loading were controlled by Western blotting using antibodies against Tublin and Lamin B. n.d., no band detected by the quantification software (see Materials and methods).

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
2.
Figure 5.

Figure 5. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

PI3K controls BAFF-induced Akt activation and B cell survival. (A) BAFF induces phosphorylation of p110. The PI3K subunit p85 was immunoprecipitated from extracts of BAFF-treated cells, and tyrosine-phosphorylated proteins in immunoprecipitates were analyzed by Western blotting using antiphosphotyrosine antibody. The amounts of p85 and p110 in the immunoprecipitates were measured by Western blot analysis. (B) PI3K inhibition suppresses BAFF-induced Akt, GSK-3β, and FoxO1 phsophorylation. B cells were stimulated with BAFF in the absence or presence of LY294002, and protein phosphorylation was measured as described in Fig. 4. (C) Inhibition of PI3K reduces survival of BAFF-treated cells. B cells were cultured in medium alone (circles) or in the presence of BAFF (squares), LY294002 (triangles), or BAFF together with LY294002 (diamonds). The frequencies of viable B cells were determined by FACS analysis.

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
3.
Figure 1.

Figure 1. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

BAFF increases size, protein content, and mitochondrial membrane potential of B cells. Data represent three independent experiments. (A) Mature B cells were incubated in culture medium in the presence of BAFF, and the cell size was measured by FACS analysis of the forward scatter (FSC). (B) Protein amount per million B cells. Error bars in A and B represent SD. (C) Mitochondrial membrane potential was measured by TMRE labeling. Histograms show the TMRE fluorescence of B cells incubated in the absence (shaded area, continuous line) or presence (open area, bold line) of BAFF. In control samples (dashed lines), membrane potential was dissipated by addition of the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone.

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
4.
Figure 2.

Figure 2. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

BAFF induces selective changes in gene transcription. The expression pattern and levels of polysome-associated mRNAs in unstimulated and BAFF-treated B cells were determined by Affymetrix microarray analysis (Table S2). The biological significance of BAFF-induced changes in gene expression was then analyzed using the GO tool GoMiner. GO categories representing biological processes are displayed as a directed acyclic graph, and those overrepresented in BAFF-stimulated cells are marked. (A) Colors indicate a statistical significance of P < 0.001 (pink) or P < 0.0001 (red). (B and C) Two prominent clusters of overrepresented GO categories are framed and displayed as 1 glycolysis and 2 cell cycle. Labeling represents GO “Biological Process” category names. Colors denote p-values as in A. Gene identities for the displayed GO categories are listed in Table S1.

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
5.
Figure 3.

Figure 3. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

BAFF induces up-regulation of cell cycle–controlling proteins but not entry into S phase. (A) Purified B cells were incubated with BAFF for the indicated times, and the expression levels of cell cycle regulatory proteins cyclin D2, cyclin E, Cdk4, Mcm2, Mcm3, Ki67, Survivin, and Rb were measured by Western blotting. Protein loading was controlled by tubulin expression level analysis. Numbers represent the fold induction normalized to the Tubulin signal. Hyperphosphorylated Rb (pRb) is identified by a migratory shift. (B) B cell proliferation in vitro was measured by BrdU incorporation. Frequencies of BrdU+ B cells incubated in medium alone (diamonds); in the presence of BAFF (closed triangles), anti-IgM (closed squares), or BAFF and anti-IgM (open triangles); or after preincubation with BAFF for 24h followed by anti-IgM treatment for the indicated times (open squares) are shown.

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
6.
Figure 6.

Figure 6. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

PKCβ controls BAFF-induced Akt phosphorylation. (A) BAFF induces membrane translocation of PKCβ. B cells were stimulated with BAFF followed by preparation of cytoplasmic and membrane protein extracts. PKCβ and Akt content in the extracts was measured by Western blotting. Fraction purity and protein loading was controlled by Western blotting using antibodies against Tubulin and Lyn. Numbers represent the fold change of PKCβ or Akt content in the membrane fractions normalized to the amount of Lyn. (B) BAFF induces PKCβ-Akt association. B cells were stimulated with BAFF, and protein extracts were treated with Akt antibody or control serum. The amounts of PKCβ or Akt in immunoprecipitates (IP) or whole cell lysates (WCL) were measured by Western blot analysis. Amounts of PKCβ in Akt-IPs were quantified as fold change over the corresponding Akt signal. (C) Akt phosphorylation levels in B cell lysates derived from control and BAFF-treated PKCβ+/+ and PKCβ-deficient mice were measured and quantified as described in Fig. 4. n.d., no band detected by the quantification software (see Materials and methods). (D) B cells from PKCβ+/+ and PKCβ-deficient B cells were stimulated with BAFF or anti-IgM antibody as indicated. Akt phosphorylation in cell lysates was measured and quantified as described in C. Because there was no band detected by the quantification software at time 0 (n.d.), numbers in D represent the total quantified area of the phosphospecific signal normalized to the total Akt content.

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
7.
Figure 4.

Figure 4. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

BAFF activates proteins required for protein translation and cell survival. (A) Purified B cells were incubated with BAFF for the indicated times, and phosphorylation of proteins that control translation was measured by Western blot analysis of eIF4E (pS209), 4E-BP1 (pS65), and S6 (pS235/236). Membranes were stripped and reprobed with nonphosphospecific antibodies against the respective proteins. Numbers represent the fold change of the phosphospecific signal normalized to the nonphosphospecific signal. (B) BAFF-induced Akt phosphorylation on S473 and T308 was measured by immunoblot analysis using the respective phosphospecific antibody (top). Akt expression was controlled by reprobing with a nonphosphospecific Akt antibody. Quantification was done as in A. (C) BAFF-induced phosphorylation of TSC2 at T1462 and of FoxO1 at S256 were measured by sequential immunoblot analysis (left). Erk is provided as a loading control to illustrate changes in FoxO1 total protein content. BAFF-induced phosphorylation of GSK-3β at S9 was measured in a similar fashion on a separate gel (right). In all cases, the amount of tested proteins was measured by reprobing the immunoblots with nonphosphospecific antibodies against the respective proteins. Quantification was done as in A. (D) BAFF induces expression of Pim-2. The positions of three different Pim-2 isoforms are indicated. Erk expression is provided as a protein loading control in C and D. n.s., nonspecific.

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.
8.
Figure 7.

Figure 7. From: BAFF controls B cell metabolic fitness through a PKC?- and Akt-dependent mechanism.

BAFF-mediated cellular responses of PKCβ-deficient B cells are altered in vitro and in vivo. (A) B cells from PKCβ+/+ (squares) and PKCβ-deficient (triangles) B cells were cultured in medium alone (open symbols) or in the presence of 25 ng/ml (light gray symbols), 100 ng/ml (dark gray symbols), or 250 ng/ml (closed symbols) of BAFF, and the frequencies of viable B cells were measured by FACS analysis. (B) The cell size of live BAFF-treated PKCβ+/+ (squares) and PKCβ-deficient (triangles) B cells was measured by FACS analysis of the forward scatter (FSC). Symbol shadings represent the same BAFF concentrations as in A. (C) BAFF-R expression on splenic B220-positive cells from PKCβ+/+ (continuous black line) and PKCβ-deficient (dashed black line) was measured by FACS. Gray lines (continuous and dashed) represent staining with an isotype control antibody. (D) Maturity of splenic B cells from PKCβ+/+ (left) and PKCβ-deficient (right) mice was assessed by expression of the surface markers IgD versus IgM (top) and CD21/35 versus IgM (bottom). Only live lymphocytes are shown, and the bottom panel is gated on B220-positve cells. Numbers in the top panels represent frequencies of cells in the respective quadrants. Gates in the bottom panels denote B cell developmental stages T1 (IgMhi CD21lo), T2 (IgMhi CD21hi), and mature (IgMint CD21int).

Alina Patke, et al. J Exp Med. 2006 October 30;203(11):2551-2562.

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