Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: FCRL3, an autoimmune susceptibility gene, has inhibitory potential on B-cell receptor-mediated signaling.

doi: 10.4049/jimmunol.0901982

Figure Lengend Snippet: FIGURE 2. Coligation of FcRIIB/FCRL3 chimeric protein inhibits BCR-induced tyrosine phosphorylation and Ca2 influx in ST486 cells. A, ST486 transfectants of chimeric proteins were stimulated with F(ab)2 of anti-IgM (10.0 g/ml) or intact anti-IgM (15 g/ml) for 3 min before analysis of whole-cell phosphorylation of tyrosine residues detected by antiphosphotyrosine Ab (PY20). Blots were striped and reprobed using anti--actin Ab. The densities of phoshotyrosine proteins were quantified and shown on the top of figure, where each transfectant stimulated with F(ab)2 anti-IgM for 3 min is set to be 1.00. B, Whole lysates from A were immunoblotted against phosphor-Syk (Tyr352) and phosoho-PLC2 (Tyr1217). Blots were striped and reprobed with anti-Syk and PLC2 Ab, respectively. The densities of phosphor-Syk and phosoho-PLC2 were quantified and shown on the top of figure, where each transfectant stimulated with F(ab)2 anti-IgM is set to be 1.00. C, Whole lysates from A were immunoprecipitated with anti-HA Ab and immunoblotted with anti-phosphotyrosine Ab. Blots were striped and reprobed with anti-HA Ab. Data are representative of four separate experiments. D, ST486 transfectants of indicated chimeric proteins were loaded with fura-2 and stimulated with 10.0 g/ml F(ab)2 of anti-IgM (solid thick line) or 15 g/ml intact anti-IgM (dotted line). Chimeric proteins were also ligated alone with biotinylated 5.0 g/ml anti-murine FcRIIB (2.4G2) followed by 10.0 g/ml streptavidin (solid thin line). The results are representative of three independent experiments.

Article Snippet: Anti-hemagglutinin (HA) Ab (12CA5) was obtained from Roche Diagnostics; anti-phospho-Syk (Tyr352) and anti-phosphoPLC 2 (Tyr1217) Abs from Cell Signaling Technologies; biotinylated antimurine Fc RIIb (2.4G2) and HRP or PE-coupled anti-phosphotyrosine Abs (PY20) from BD Biosciences; anti-PLC 2, anti-SHP-1, anti-SHP-2, anti-SHIP and agarose-conjugated anti-HA Abs from Santa Cruz Biotechnology; anti-ZAP70 Ab from eBioscience, and intact and F(ab )2 of rabbit anti-human IgM and goat anti-chicken IgY from Jackson ImmunoResearch Laboratories.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation

Journal: Acta Neuropathologica Communications

Article Title: Trichuris suis induces human non-classical patrolling monocytes via the mannose receptor and PKC: implications for multiple sclerosis

doi: 10.1186/s40478-015-0223-1

Figure Lengend Snippet: PKC signals downstream upon TsSP-MR interaction. Quantification of flow cytometry analysis of the expression levels of ( a ) CCR2, ( b ) LFA-1 avidity and ( c ) LFA-1 affinity on human monocytes. These cells were cultured in the presence or absence of TsSP (40 μg/ml, 16 h), the PKC inhibitor Bisindolylmaleimide I (GF109203, 2 μM) and blocking antibodies for the MR or its isotype control (both 10 μg/ml). ( b ) and ( c ) are expressed as the percentage of avidity/affinity compared to total LFA-1. Cell supernatants were used to determine the levels of ( d ) TNF-α and ( e ) IL-10 secretion by enzyme-linked immunosorbent assay analysis. f PKC activation was determined by Western Blotting using phosphoPKC antibodies compared to GAPDH expression and images were subsequently quantified using ImageJ. Experiments were performed in triplicate using cells derived from 4 different human donors and the results are presented as the mean ( a-c ); the mean percentage ( d, e ) or the mean PKC phosphorylation +/− SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by ANOVA and Students t test

Article Snippet: The following primary antibodies (in TSM/0,05 % Tween/5 % BSA) were used: PhosphoPKC (Cell signaling, Beverly, MA, USA, #9371S) and GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-32233) as a loading control.

Techniques: Flow Cytometry, Expressing, Cell Culture, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Derivative Assay, Phospho-proteomics