1. RhoA GTPase Regulates B Cell Receptor Signaling
Abdelhafid Saci, Christopher L. Carpenter 
Molecular Cell 
Volume 17, Issue 2, Pages (January 2005)
DOI: /j.molcel

2. Figure 1 RhoA Is Activated following BCR Stimulation
A20 (A) or primary splenic B cells (B) were starved and then stimulated via the BCR for 2, 5, or 10 min with F(ab)′2, in the presence or absence of 100 nM wortmannin. Cells were lysed and centrifuged, and then GST-RBD bound to GSH beads were incubated with the soluble fraction. Samples were subjected to SDS-PAGE and Western blotting using an anti-RhoA antibody (upper panels, GST-RBD). Aliquots from each lysate were also Western blotted to verify the amount of RhoA in each condition (lower panels, lysate). These results are representative of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 RhoA Regulates BCR-Dependent Ca2+ Flux in A20 Cells
(A) A20 cells were infected with either wild-type vaccinia virus (pSC65) or a virus expressing V14RhoA or N19RhoA. Cells were then loaded with Fura-2, the BCR was stimulated with 15 μg/ml F(ab)′2, and the Fura-2 fluorescence ratio was determined.
(B) A20 cells were infected by pSC65 or by N17Cdc42 recombinant vaccinia virus, and then calcium flux was measured as in (A). The insets represent the expression of RhoA mutants (A) and N17Cdc42 (B) tested with Western blotting before adding Fura-2.
(C) A20 cells were treated or not with C3 toxin before loading with Fura-2, and calcium flux was measured as in (A).
(D) A20 cells were infected and treated as in (A) and then stimulated with a submaximal concentration (2 μg/ml) of F(ab)′2.
(E) Infected primary B cells were loaded with Fura-2 and stimulated with 20 μg/ml F(ab)′2, and calcium flux was measured.
(F) Primary B cells were infected with the indicated viruses and then lysed and subjected to SDS-PAGE and Western blot analysis with anti-myc antibody to verify RhoA mutant expression. These results are representative of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
RhoA Regulates BCR-Dependent IP3 Production, but Not PLCγ2 or Akt Phosphorylation
(A) A20 cells were infected with the indicated vaccinia viruses and then loaded with Fura-2 and resuspended in calcium-free buffer containing 2 mM EGTA. The BCR was then stimulated, and calcium flux was measured.
(B) A20 cells infected with the indicated viruses were stimulated with F(ab)′2, and after 2 min the cells were lysed and IP3 production was measured as described in the Experimental Procedures.
(C and D) Infected A20 cells were stimulated or not through the BCR (30 μg/ml F(ab)′2) and lysed, and phosphorylation of PLCγ2 (C) or of Akt (D) was analyzed by Western blot. Total PLCγ2 and Akt were also determined (lower panels). Results are representative of three experiments.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 RhoA Regulates BCR-Dependent Phospholipid Synthesis
(A) A20 B cells (left) or primary splenic B cells (right) were infected with recombinant vaccinia viruses and then were incubated in phosphate- and serum-free medium for 1–2 hr. Cells were then labeled with 32P-PO4 for 5 min at 37°C and stimulated for 3 min. The reaction was stopped with 1 N HCl, and lipids were extracted with methanol/chloroform (1:1) and separated by TLC (thin-layer chromatography). Phospholipid synthesis (PtdIns-4,5-P2, PtdIns-4-P and PA) in A20 cells was quantified using a PhosphorImager (B). These results are representative of four experiments.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
C3 Toxin Inhibits BCR-Dependent Phospholipid Synthesis, and RhoA Associates with Lipid Kinases
(A) A20 cells were grown in a six-well plate at 50% confluence on the day of the experiment. C3 toxin was introduced using a cell-permeable peptide, as described in the Experimental Procedures. Cells were then loaded with 32P-PO4 for 5 min at 37°C in phosphate- and serum-free medium and stimulated for 3 min. Phospholipids were extracted and analyzed as in Figure 4.
(B) Phospholipid synthesis was quantified using a PhosphorImager.
(C) A20 cell lysates were incubated with GST or GST-RhoA (loaded with GDPβS or GTPγS) bound to GSH beads for 1 hr at 4°C. The beads were washed and assayed for associated phospholipid kinase activities. RhoA or nonimmune immunoprecipitates from quiescent or stimulated A20 cells were also assayed for associated phosholipid kinase activities. Results are representative of four experiments.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
PtdIns-4,5-P2 Rescues Calcium Flux Inhibited by N19RhoA Expression
(A) A20 cells were infected with vaccinia virus expressing N19RhoA mutant or empty vector (pSC65). Cells were then loaded with Fura-2, the BCR was stimulated [15 μg/ml F(ab)′2], and calcium flux was measured.
(B) A20 cells were infected and loaded with Fura-2 as above. PtdIns-4,5-P2-histone H1 complexes were added 1 min before BCR stimulation, and calcium flux was measured.
(C) A20 cells were infected and loaded with Fura-2 as above. Either PtdIns-4,5-P2-histone H1 or PtdIns-3,5-P2-histone H1 complexes were added 1 min before BCR stimulation, and calcium flux was measured. Upward arrows indicate BCR stimulation, and downward arrows indicate where lipids were added. Results are representative of four experiments.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 RhoA Regulates BCR-Dependent Cell Proliferation
(A) Mouse splenic B cells were either treated with the Chariot peptide alone or transduced with 5 μg/ml C3 toxin for 3 hr using the peptide. Cells were stimulated in duplicate with either rabbit anti-mouse IgM F(ab)′2 antibody (10 μg/ml) or LPS (10 μg/ml) for 40 hr in the presence of [3H]thymidine for the last 18 hr.
(B) After C3 toxin transduction by Chariot, cells were loaded with Fura-2 and subjected to calcium flux analysis upon stimulation with 10 μg/ml anti-IgM F(ab)′2 antibody. These results are representative of four experiments.
Molecular Cell  , DOI: ( /j.molcel )