Supplemental Material to: EMBO J. 28: 711

Supplemental Figure 1: Purification of lipid rafts and assessment of Bright levels in sub-cellular compartments.

(A) Isolation and purity of lipid rafts. Raji, Ramos, CL01 and Daudi (10⁷ cells) were subjected to preparation of plasma membranes (M) and lipid rafts (R) as described in Material and Methods. Fractions were taken from the top of the gradient to the bottom and analyzed by Western blot using α-Raftlin anti-serum (upper panel). Whole cell lysate (WCL) from HeLa cells served as control. Filters were probed with antibodies against Golgi- (GM-130; Short and Barr, 2000) and nuclear- (Lamin-B; Shelton et al, 1982) membrane markers (lower panel).

(B) Biochemical subcellular fractionation and semi-quantitative measurement of Bright levels. Exponentially growing Daudi and CL01 cells were fractionated into cytoplasm (CY), soluble nucleoplasm (NP), chromatin (CH) and nuclear matrix (NM) according to previously published protocols (Kim and Tucker, 2006; Liu et al, 2006). Immunoblotting of equal aliquots (~25 µg) of protein with α-Bright anti-serum (lanes 1 to 8) revealed no significant differences between these two cell lines. The cytoplasmic extracts (lanes 1, 9, 5 and 12; 25mg as an equivalent to ~2x10⁶ cells) were used to normalize the Western (lanes 9 to 14). Plasma membranes (M; lanes 10 and 13; Raftlin negative) and lipid rafts (R; lanes 11 and 14; Raftlin positive) from ~10⁷ cells were separated using the flotation method, as described in the Methods section. The entire preparation was used for SDS-PAGE/Western, which was internally normalized by re-probing the filter with α-Raftlin anti-serum (Raflin provided an optimal loading control because lipid rafts prepared from either Daudi or CL01 cells exhibit indistinguishable levels of Raftlin; data not shown).

Supplemental Figure 2: Human B cell lines and primary human peripheral CD43- B cells have different BCR thresholds.

(A) Raji B cells are more sensitive to low levels of anti-IgM stimulation than Ramos cells. Raji is an EBV-positive lymphoblastoid cell line expressing LMP2A (Caldwell et al, 1998; Bernasconi et al, 2006), which is ascribed to down-regulate BCR signaling (as an example, please see published reports McConnell et al [1992] and Miller et al [1993] for a range of concentrations used). We employed a dose-response of exponentially growing Raji and Ramos cells (5x10⁸ cells) to 5 min treatment of varying concentrations of anti-IgM F(ab')₂ (clone JDC-150), followed by analysis of whole cell lysates using antibodies against phosphorylated tyrosine (pY) and γ-Tubulin as a loading control. Consistent with the studies cited above and numerous others, higher concentrations of stimulating F(ab')₂ fragment yielded a far more robust phosphotyrosine footprint for EBV-negative Ramos cells (lanes 9 and 10) than for Raji cells (lanes 4 and 5). However, at lower concentrations (Materials and Methods) of F(ab')₂ employed in all our signaling studies (except Supplemental Figure 4B)_, Raji cells were more susceptible to BCR stimulation (lanes 2 and 3) than were Ramos cells (lanes 7 and 8).

(B) Differential responses of B cell lines to weak (anti-IgM) or strong (anti-IgM + α-CD19) BCR engagement correlates with levels of Bright within their lipid rafts. The indicated cells (~5x10⁸) were stimulated for stimulated for 5 min using either 500ng α-μ or 500ng α-μ + 500ng a-CD19. Whole cell extracts were prepared, and aliqouts were analyzed by Western blot using α-phosphotyrosine mAb (p-Y) and α-Lamin (loading control).

(C) Raji and Daudi B cells undergo stronger apoptotic responses to long-term BCR stimulation. The indicated cells (~1x10⁸) were stimulated for 72 hr with 100ng α-μ. DNA fragmentation patterns were assessed by ethidium bromide staining of gel electrophoresed DNA.

(D) Primary human peripheral B cells display a signal strength-dependent discharge of Bright from lipid rafts and accumulation of Sumo-I-Bright in plasma membranes.Human CD43^- PBL (~2x10⁶) were stimulated for 5 min using 2ng a-m or 2ng a-CD19, or 2ng a-m + 2ng a-CD19, as indicated at the top of each panel. Upper panel, lipid rafts prepared from ~10⁶ cells per lane were subjected to immunoprecipitation by anti-Bright, then western blotted with anti-Bright and anti-Sumo-I antibodies; 10% of the input was probed with anti-IgM (μ) to confirm imunoprecipitate input for lane 4. Lower panel, whole cell lysates (WCL, prepared from ~10⁶ cells per lane) were immunoblotted with anti-phosphotyrosine (p-Y), anti-Bright and anti-g-tubulin (loading control).

(E) RIPA is sufficient to solubilize lipid raft for co-immunoprecipitation. Inherent dynamics of lipid rafts and their resistance to ice-cold detergents can lead to a detection artifact of protein aggregates instead of protein-protein complexes, due to the unique native structure of these platforms for BCR signaling (Dintzis et al, 1976; Cheruki et al, 2001; Sproul et al, 2000; Putnam et al, 2003; Saeki et al, 2003; Gupta et al, 2006; Depoil et al, 2008). To control for solubilization effects on our immunoprecipitation studies presented in Figure 2B, exponentially growing Daudi cells (~5x10⁸) were subjected to preparation of lipid rafts, as described in the Methods section. Fractions were pooled and treated with RIPA (500mM NaCl; 10mM Tris/Cl pH 8; 0.1% SDS; 5mM EDTA, pH 8; 10x protease inhibitor [Complete tablet, Roche]; 15min on ice) as indicated in the figure. Samples were then subjected to IP/Western analysis using the antibodies indicated. While a complex between μ and Raftlin is sensitive to RIPA treatment (in agreement with Saeki et al, 2003), we found that a complex containing Raftlin and Btk is precipitable regardless of RIPA treatment.

Supplemental Figure 3: Bright is Sumo-I modified.

(A) Bright associates with Sumo-I E2 (PIAS1) and E3 (PIAS1) conjugating enzymes. To search for potential interaction partners of Bright, a human buffy coat-derived, GAL4-Activation domain-fusion cDNA library (Clontech) was screened with a full-length GAL4-DNA binding domain fusion of Bright (data not shown). Potential interacting partners were colony purified and further analyzed. Insert (top): growth of yeast clones, expressing both Bright and PIAS1 encoding plasmids. Increasing concentrations of 3-amino-triazole (3-AT), a competitive inhibitor of the imidazole glycerolphosphate dehydratase, was used to determine the strength of the protein-protein-association. Panel (below): A semi-quantitative liquid GAL4 assay was used to determine specificity and strength of complexes among Bright, Ubc9 and PIAS1. The published Bright-SP100 interaction was used as internal control (Zhong et al, 2000).

(B) Sumo-I modification of Bright in vivo and in vitro is sensitive to Sumo-I peptidases (Ulp-1 and Senp1) but enhanced by Sumo-I E1 and E2 (Sae2/1 and Ubc9). Lanes 1-3: GFP-Bright (lanes 1-3) was co-expressed with GFP-Sumo1 (lanes 2 and 3) and Ulp-1 (lane 3) in Cos-7 cells. RIPA whole cell lysates from 10⁶ cells per lane were analyzed by IP/Western using α-Bright anti-serum. The arrow (lane 2) indicates the putative Sumo-I-Bright conjugated species of lower electrophoretic mobility. Specificity of the sumoylation reaction was confirmed by cleavage of modified Bright by the Sumo-I iso-peptidase, Ulp-1 (lane 3; Li and Hochstrasser, 2003) Lanes 4-6: V5 epitope-tagged Bright (lanes 4-6) was in vitro translated and incubated (as indicated) with purified E1 (Sae2/1), E2 (Ubc9) and Sumo-I as described (Rosas-Acosta et al, 2005a,b). Sumo-I-conjugated species are marked with arrows in lane 6. Lanes 7-10: Cos-7 cells were transiently transfected with the constructs indicated (lanes 7 to 10) and 48 hr later, whole cell extracts from 10⁶ cells per lane were prepared in the presence of 200mM iodoacetic acid sodium salt in RIPA buffer (Byrd and Hruby, 2005; Bossis and Melchior, 2006). Unmodified and modified Bright were identified by anti-Bright western. As with the majority of sumoylated proteins, Sumo-I-Bright was stabilized against Sumo-I hydrolases by alkylation and degraded by over-expression of the Sumo-I-specific hydrolase, Senp1 (lane 7; Rosas-Acosta et al, 2005a,b). The size and laddering of Sumo-I-Bright (the range is indicated by a square bracket) suggested that, when fully stabilized, Bright is poly-sumoylated (lanes 9 and 10; see Gocke et al [2005] for the nomenclature). Lanes 11-14: Cos-7 cells were transiently transfected with wild-type or Sumo-I mutant ⁴⁰¹KIKK /AIAA forms of Bright, subjected to preparation of whole cell lysates (10⁶ cells per lane using RIPA supplemented with 200mM iodoacetic acid). Immunoprecipitation was performed with anti-Sumo-I followed by an anti-Bright western. No effects of Sumo-I protease or alkylation were observed on the Sumo-deficient mutant (lanes 11, 12), or in similarly prepared extracts from CL01 B cells (lanes 13 and 14). The molecular weight range of Sumo-I-Bright species (indicated by a square bracket) suggests that 2-3 chains of Sumo-I are conjugated at ⁴⁰¹KIKK.

(C) In the absence of BCR stimulation, Sumo-I modified Bright partitions to the nucleus and to the cytoplasm, but not into lipid rafts. Upper panel: Resting Daudi cells (10⁸) were fractionated into whole cell extracts (W), lipid rafts (R), cytoplasm (C) and nuclear proteins (N), followed by IP/Western analysis using the indicated antibodies. Regardless of whether IP is performed with α-Bright or with α-Sumo-I, the sumoylated form of Bright co-migrates with the smaller, faster-migrating and predominant species; the composition of the upper species likely corresponds to an oxidized form of the single cysteine residue, C342. Lower panel: Western analysis of 1% of the input, used for the above immunoprecipitation assay, probed with anti-Bright. Purity of the fractionation is demonstrated by probing with antibodies against Raftlin, G6PDH and Histone-H₁.

(D) Wild-type and Sumo-1-deficient Bright accumulate in lipid rafts and membranes of fibroblasts. NIH/3T3 fibroblasts were transiently transfected with the indicated expression constructs. Plasma membranes and lipid rafts were prepared from 10⁶ cells and analyzed by IP/Western using α-Bright.

Supplemental Figure 4: Dominant negative forms of of Bright complex with endogenous Bright and alter BCR signaling threshold.

(A) Endogenous Sumo-I-Bright pull-down down using V5 tagged ⁴⁰¹KIKK/AIAA-Bright. Raji cells (~5x10⁸), stably expressing a V5-tagged Sumo-I-mutant form of Bright (⁴⁰¹KIKK /AIAA) and empty vector controls were homogenized in RIPA/iodoacetic acid, immunoprecipitated with anti-Sumo-I antibody (which would only detect endogenous Sumo-I-modified Bright), and then western blotted with anti-V5 (which would only detect ectopically delivered ⁴⁰¹KIKK /AIAA-Bright) or with anti-Bright (detects both endogenous and exogenous Bright).

(B) Phosphotyrosine footprint of Raji cells, stably expressing wild-type and mutant forms of Bright, in response to increasing amounts of α-μ. Raji (~2x10⁶) cells, stably expressing the indicated forms of Bright, were subjected to 5 min stimulations using 2ng (low), 1μg (medium) or 80μg α-μ (high), followed by analysis of whole cell extracts and lipid rafts using the antibodies indicated; the equivalent of 10⁶ cells per lane is loaded.

Supplemental Figure 5: Transgenic over-expression of Bright alters BCR signaling and co-localization with membrane IgM.

(A) Response of wild-type and Bright transgenic CD43^- B cells to increasing amounts of BCR ligation with anti-IgM. CD43^- splenic B cells (~2x10⁶) were prepared via negative selection from wild-type and Bright transgenic mice, and then were subjected to 5 min stimulations using 2ng (low), 1μg (medium) or 80μg (high) doses of F(ab^’)₂ anti-IgM as indicated by triangles. Whole cell extracts (WCL) and lipid rafts (~10⁶ cells/lane) were analyzed by western using the antibodies indicated. Saturation of the pTyr response in wild type cells was achieved at the high dose (80μg) condition. The ratio of signal achieved at low (lane 6) vs high (lane 8) anti-IgM dose, along with mean fluorescence intensities measured by FACS (data not shown) indicated that ~1-5% of mIgM was ligated in wild type B cells following stimulation with low dose (2ng) anti-IgM.

(B) Bright co-localization with mIgM is greater in MZB than in FO B cells. Purified wild-type and Bright over-expressing transgenic FOB and MZB cells (see Figure 6B for details) were stimulated for 5 min using 1pg a-m + 1pg a-CD19 for 10³ cells, fixed and stained with α-Bright, α-μ and the DNA dye, Hoechst. Images were collected as described in Materials and Methods. Co-localization (yellow) between Bright (red) and IgM (green) is indicated by arrowheads and was confirmed by deconvolution imaging (movies are available upon request).

Supplemental Video 1: Bright and mIgM co-localize in resting mouse CD43^- splenocytes. Mouse CD43^- splenocytes were immunostained for mIgM, Bright and DNA. Image stacks were deconvolved for 500 iterations using the expectation maximization algorithm in XCOSM, and 3D projections were calculated using the maximum value projection method in ImageJ. The three channels have been combined in a pseudocolor image with Bright in red, mIgM in green and DNA in blue.

Supplemental Video 2: Bright and mIgM co-localization is lost in α-μ stimulated mouse CD43^- splenocytes. Mouse CD43^- splenocytes were stimulated for 5 min using 1pg a-m for 10³ cells, followed by immunostaining of mIgM, Bright and DNA. Image stacks were deconvolved for 500 iterations using the expectation maximization algorithm in XCOSM, and 3D projections were calculated using the maximum value projection method in ImageJ. The three channels have been combined in a pseudocolor image with Bright in red, mIgM in green and DNA in blue.