Basic and translational research Figure 3 CHiP-qtPCR showing binding of NFκB p50 as well as NFκBp65 within the genomic segment contained in the 1.2 Kb haplotype window in Daudi cells. (A) mRNA expression of the BLK gene after BcR stimulation. (B) qtPCR of the portion containing SNP rs2248932 within the 1.2 kb genomic segment in intron 1 of human BLK. (C) qtPCR of the IkBa gene used as positive control. CHiP-qPCR for NFkB p50 and p65 on the 1.2 Kb associated haplotype genomic segment CHiP experiments were performed according to the manufac- turer’s recommendations (EZ-Magna CHiP A, Millipore, Billerica, California, USA). In brief, 4×10−6 Daudi cells were incubated with or without 10 mg/ml anti-immunoglobulin M (IgM) F(ab′)2 fragments (Jackson Immunoresearch, West Grove, Pennsylvania, USA) for the indicated time. Cells were ﬁxed with 1% formal- dehyde and nuclei were isolated and sonicated within 300 ml of lysis buffer. An amount of 50 ml of chromatin–protein complexes were immunoprecipitated overnight at 4°C by mild agitation with 5 mg of pAb speciﬁc for p65 (Santa Cruz, California, USA) or p50 (Abcam, Cambridge, Massachusetts, USA) or normal rabbit IgG (Millipore) as negative control. After elution from the immunopre- cipitated chromatin complexes followed by reverse-crosslink and cleanup, DNA samples were subjected to real-time PCR analysis using primers as follows: rs2248932 neighbouring region within Blk intron 1 primer set: forward – TGCATCAGCATCACTGGG; reverse – GGCACAATAGTTCTGAAACCTC. IkBa was used as control for NFkB binding with the following primer set: forward – CACTTGCAGAGGGACAGGAT; reverse – GAGAAACTCCCTGCGATGAG. Cloning and expression of BLK constructs with the Ala71Thr substitution BLK sequence was PCR ampliﬁed using cDNA from a BJAB cell line and primers f-BLK: 5′-CACCatggggctggtaagtagc-3 and r-BLK 5′-gggctgcagctcgtactgcc-3. The start codon in the forward primer is underlined. The open reading frame was 1222 cloned in pcDNA3.1D/V5-His (Invitrogen, Life Technologies, Grand Island, New York, USA) and conﬁrmed by sequenc- ing. The protein tagged with the V5 epitope at the C terminal was produced by deletion of the stop codons. The BLK 71Thr isoform was produced by mutagenesis PCR with pfu-Ultra II fusion HS polymerase (Stratagene, Agilent Technologies, Santa Barbara, California, USA) and the primers BLKm71f: 5′-TATGACTACACCaCTATGAATGATCGGGACCT-3 and BLKm71r: 5′-ATCATTCATAGtGGTGTAGTCATACAGAG-3. Expression of the constructs was assayed by western blot with anti-V5 antibody (online ﬁgure S4). Protein stability assay Embryonic kidney HEK293T cells grown in RPMI 1640 medium supplemented with 10% fetal calf serum were seeded on six- well plates and transfected with 4 μg of expression plasmids V5-tagged BLK isoforms using Lipofectamine 2000. At 22 h cells were harvested and distributed in six wells of 12-well plates. After 2 h cells were treated with 20 μg/ml cycloheximide in dim- ethyl sulphoxide (DMSO) or DMSO alone (time 0) for various times. Cellular extracts were prepared in lysis buffer: 1% NP40, 150 mM NaCl, 10 mM Tris (pH 7.5), 2 mM sodium orthova- nadate, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfo- nyl ﬂuoride and protease and phosphatases inhibitor cocktails (Roche). A ﬁfth of the extract was resolved on SDS-PAGE and immmunoblotting carried out using standard protocols with anti-V5 antibodies (Invitrogen), b-tubulin (Sigma-Aldrich) and antimouse IgG-HRP (Zymed, San Francisco, California, USA). Quantiﬁcation of the bands was done using ImageJ. Ann Rheum Dis 2012;71:1219–1226. doi:10.1136/annrheumdis-2011-200987