Specific SHIP1 activity measurement.

<p>(A) Specificity of the assay was determined by immunoprecipitation (IP) of SHIP1 from multiple myeloma cell lines (U266 and OPM2) that express SHIP1 (see Western blot insert of whole cell lysates) and breast cancer cell line (MCF7) that does not. Phosphatase activity of immunoprecipitates was determined by adding the SHIP1 substrate PtdIns(3,4,5)P₃, followed by Malachite Green reaction. Human recombinant SHIP2 was used as positive control for the phosphatase assay. Only immunoprecipitates from SHIP1-competent cells show phosphatase activity. (B) OCRL1, SHIP2 or SHIP1 were immunoprecipitated from SHIP1-competent U266 cells and SHIP1-negative MDA-MB-231 (MDA) breast cancer cells. IPs were run on Western blot and probed for SHIP1, SHIP2 and OCRL1 (upper panel). Whole cell lysates (WCL) were also subjected to Western blot analysis of these phosphatases (lower panels). OCRL1 and SHIP2 were precipitated from both MDA-MB-231 and OPM2 cells, whereas SHIP1 was only observed in OPM2, emphasizing specificity of the IP. (C) Malachite Green phosphatase assay (wells shown in lower panels) shows no activity in SHIP1 precipitates from MDA-MB-231 cells, whereas SHIP1 activity was observed in U266 cells. In contrast, OCRL1 phosphatase activity was detected in both cell lines.</p

Patient characteristics (patients included in the SHIP activity assay).

<p>Patient characteristics (patients included in the SHIP activity assay).</p

Aberrant SHIP1 activity and expression in adult CD patients.

<p>(A) PBMCs from CD patients and healthy controls (HC) were lysed, phosphatase assay performed. Examples of 3 of 5 CD patients with abrogated SHIP1 expression are shown. Lower panels show SHIP1, PTEN and actin protein in the same PBMC lysates of CD patients and healthy controls. Phosphatase activity in these patients was tested in duplicate, with identical results. (B) Correction of SHIP1 phosphatase activity for the amount of SHIP1 protein in the lysates shows that intrinsic SHIP1 enzymatic activity is significantly lower in CD patients compared to HC. (C) Increased mRNA and protein (examples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182308#pone.0182308.s003" target="_blank">S2A Fig</a>) expression compensates for the reduced intrinsic enzymatic activity (CD patients [n = 47] vs healthy controls [n = 42], 1.68 fold increase, p = 0.0042). RNA was available of three of the five SHIP1-deficient patients (open circles). (C) PTEN protein expression in PBMC lysates from CD patients and healthy controls (HC) were determined by Western blot analysis, and corrected for Actin levels in the same samples. Quantification of individual experiments, including mean ±SD are shown. (D,E) PTEN (D) and SHIP2 (E) protein expression in PBMC lysates from CD patients and healthy controls (HC) were determined by Western blot analysis, and corrected for Actin levels in the same samples. Quantification of individual experiments, including mean ±SD are shown (examples in Fig 2A lower panel and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182308#pone.0182308.s003" target="_blank">S2B Fig</a>).</p

Specific SHIP1 activity measurement.

<p>(A) Specificity of the assay was determined by immunoprecipitation (IP) of SHIP1 from multiple myeloma cell lines (U266 and OPM2) that express SHIP1 (see Western blot insert of whole cell lysates) and breast cancer cell line (MCF7) that does not. Phosphatase activity of immunoprecipitates was determined by adding the SHIP1 substrate PtdIns(3,4,5)P₃, followed by Malachite Green reaction. Human recombinant SHIP2 was used as positive control for the phosphatase assay. Only immunoprecipitates from SHIP1-competent cells show phosphatase activity. (B) OCRL1, SHIP2 or SHIP1 were immunoprecipitated from SHIP1-competent U266 cells and SHIP1-negative MDA-MB-231 (MDA) breast cancer cells. IPs were run on Western blot and probed for SHIP1, SHIP2 and OCRL1 (upper panel). Whole cell lysates (WCL) were also subjected to Western blot analysis of these phosphatases (lower panels). OCRL1 and SHIP2 were precipitated from both MDA-MB-231 and OPM2 cells, whereas SHIP1 was only observed in OPM2, emphasizing specificity of the IP. (C) Malachite Green phosphatase assay (wells shown in lower panels) shows no activity in SHIP1 precipitates from MDA-MB-231 cells, whereas SHIP1 activity was observed in U266 cells. In contrast, OCRL1 phosphatase activity was detected in both cell lines.</p

SHIP1 expression and activity are independent of <i>ATG16L1</i> SNP status.

<p>ATG16L1 rs2241880 SNP status was determined for those subjects for whom genomic DNA was available. Groups were stratified according to protective (AA), heterozygous (AG) or risk (GG) alleles. (A) SHIP1 activity corrected for total amount of SHIP1 present in the lysates is shown. The number of risk alleles does not affect SHIP1 intrinsic activity levels. (B) SHIP1 mRNA levels corrected for RPLP1 mRNA levels are shown for patients and controls, stratified according to <i>ATG16L1</i> SNP status. The number of <i>ATG16L1</i> risk alleles does not affect SHIP1 mRNA expression.</p