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Blood, Vol. 113, Issue 5, 1037-1044, January 29, 2009
Critical role of class IA PI3K for c-Rel expression in B lymphocytes
Blood Matsuda et al.
113: 1037
Supplemental materials for: Matsuda et al
Files in this Data Supplement:
- Figure S1. Phenotypes of p85α−∕− B cells on a C57BL/6 background (JPG, 79.7 KB)
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(A) Splenocytes from the indicated mice were analyzed for B220 and AA4.1 expression on a FACSAria. The percentages of immature B cells (B220+AA4.1+) and mature B cells (B220+AA4.1−) are indicated. (B) Analysis of B220+AA4.1+ immature B cells for surface expression of IgM and CD23: T1, IgM+CD23lo; T2, IgM+CD23+; T3, IgMloCD23+. Among B220+AA4.1+ cells, the percentages of the T1, T2, and T3 subsets are indicated. Data are representative of three mice.
- Figure S2. Phenotypes of p85α−∕− T cells on a C57BL/6 background (JPG, 44.8 KB)
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Splenic CD4+ T cells from the indicated aged mice (5-month-old) were analyzed for CD44 and CD62L expression on a FACSAria. The percentage of naïve T cells (CD44−CD62L+) and memory T cells (CD44+CD62L−) are indicated. Data are representative of three mice for each genoptype.
- Figure S3. PI3K is dispensable for PLCγ2 and PKCβ activation (JPG, 34.1 KB)
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(A) Splenic B cells obtained from wild-type mice were stimulated with or without 20 µg/ml of anti-IgM F(ab′)2 (anti-µ) along with or without 10 µM LY294002 (Calbiochem) at 37°C for 3 min. PLCγ2 was then immunoprecipitated with an anti-PLCγ2 antibody (SantaCruz), followed by immunoblot analysis with 4G10 (pPLCγ2). The membrane was re-blotted with the anti-PLCγ2 antibody (PLCγ2). (B) Splenic B cells obtained from the indicated mice on a BALB/c background were stimulated with or without 20 µg/ml of anti-IgM F(ab′)2 (anti-µ) at 37°C for 3 min. Btk was then immunoprecipitated with an anti-Btk antibody, followed by immunoblot analysis with a specific antibody (kindly provided by Dr. D. Rawlings) recognizing Btk phosphorylated at Ser 180 (pBtk (S180)). The membrane was re-blotted with 43-3B (Btk). Data are representative of two independent experiments with similar results.
- Figure S4. Comparison of c-Rel expression levels between p85α+∕− and p85α−∕− mature B cells (JPG, 22.7 KB)
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(A) Splenic B cells obtained from the indicated mice on a BALB/c background were stained with B220 and AA4.1, and B220+AA4.1− mature B cells were sorted on a FACSAria (purity > 90%). The cells were then subjected to immunoblot analysis for c-Rel expression, followed by re-blot with an antibody against α-tubulin (Sigma). (B) The whole cell lysates of splenic B cells obtained from CBA or Xid mice were subjected to immunoblot analysis with antibodies against c-Rel and ERK2.
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