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Figure S1. Phenotype of pre-B cell lines and sorting method for isolating precursor B cells from human bone marrow mononuclear cells. (A) 697 and Nalm6 cell lines were quantified for their surface markers (VpreB, IgM, Igβ, CD19, Igκ and Igλ) to determine their purity. (B) Mononuclear cells from human bone marrow were stained with anti-CD19 APC, -CD34 PE-Cy7, and -κ/-λ FITC. (C) The precursor B cells (CD19+, CD34lo, κ and λ) were sorted within the lymphocyte gate using FACS Aria II sorter. Sorted cells were quantified for surface VpreB and µHC expression. Filled peak represents VpreB+ orµHC cell population.

Figure S2. Pre-BCR-dependent cell cycle entry and proliferation. Six hundred and ninety seven cells were treated with anti-µHC F(ab')2 or control F(ab')2 antibodies in presence of 3% FCS. Cell cycle analysis showed increased number of cells in S-phase. (A) Trypan blue based cell count showed increased cell growth in pre-BCR stimulated cells compared to control cells. (B) Stimulation of pre-BCR on serum starved cells resulted in phosphorylation of p21Cip1 in 2 min and increase in c-Myc level after 24 h. (C) Serum starved 697 cells were treated with anti-µHC or/and anti-CD19 for 15 min or 48 h. The cell lysates were resolved on SDS–PAGE and blotted with antibodies against phospho-Rb, p27Kip1 (D), phospho-AKT (E) or phospho-ERK1/2 (F). Actin detection was used as a loading control. The blots represent one of two independent experiments.

Figure S3. Pre-BCR associated signalosome. Serum deprived 697 or Nalm6 cells were incubated with anti-µHC or control F(ab')2 antibodies or only medium (med) for various time points as indicated (top rows). Phosphorylated forms of various signalling molecules were detected using western blot after pre-BCR crosslinking. (A) LYN and VAV was immunoprecipitated using respective specific antibodies and blotted using anti-phospotyrosine (anti-p-Tyr; 4G10) antibody. BLK, PLCγ-2 and LAB were immunoprecipitated using anti-p-Tyr and blotted using respective specific antibodies. To detect phosphorylated form of SYK, total cell lysate was blotted using phospho-specific antibody. BLNK was immunoprecipitated from the cell lysate and blotted using anti-p-BLNK antibody. (B) Total cell lysates were blotted using respective antibodies for phosphorylated ZAP70, LAT and SLP76. (C) Total cell lysates were blotted using anti-p-AKT, -GSK-3β and -FKHRL1 antibodies. (D) Pre-BCR-induced Ras activation was analysed using pull-down experiments as described in Material and Methods Section. Total lysates of 697 was blotted for ERK and p38 using respective phosphospecific antibodies. Non-phosphorylated forms of all signalling molecules were also detected by re-probing with respective antibodies as a loading control. The blots shown represent one of two independent experiments.

Figure S4. Pre-BCR-induced activation of c-Cbl and its interaction with Zap70. (A and B) After the stimulation of pre-BCR by anti-µHC F(ab')2 antibodies, the cell lysates (697 or Nalm6) were blotted for detecting c-Cbl (Y774 and Y731). (C) 697 cell lysates were also submitted to immunoprecipitation with anti-ZAP70 Abs. Precipitates were resolved on SDS–PAGE and analysed by western blot for expression of c-Cbl and ZAP70. Data represented is one of two independent experiments. Actin detection was used as a loading control.

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