Anti igm antibody

The phosphorylation assay of proteins was measured by FACS with BD™ Phosflow technology (BD Biosciences) according to the manufacturer’s instructions. The stimulations for splenic B cells were examined by anti-IgM antibody (final concentration 10 μg/ml; Jackson Immunoresearch) or lipopolysaccharide (LPS, final concentration 20 μg/ml; Sigma-Aldrich, San Francisco, CA, USA) in time course of 0, 5, 10, and 15 min. In the case of IgM stimulation, the antibody set of Alexa Fluor® 488 Mouse ERK1/2 (pT202/pY204) (Erk1/2) and PE Mouse anti-Syk (pY348) (Syk) was applied to detect the phosphorylated proteins. In the case of LPS stimulation, Erk1/2 and PE Mouse p38 MAPK (pT180/pY182) (P38) (BD™ Phosflow, BD Biosciences) were examined. These antibodies with anti-B220 were stained for splenic B cells for 30 min in Phosflow experiment process.

Primary CLL cells treated with ibrutinib and/or cobicistat were stimulated by spinning onto a 6-well plate coated with anti-IgM antibody (Jackson ImmunoResearch Laboratories) at a concentration of 10 μg/mL. After 15 minutes of stimulation, cell lysates were collected and analyzed by SDS-PAGE. Protein was transferred to nitrocellulose and blots were probed with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. Blots were imaged using WesternBright chemiluminescent substrate (Advansta) on an X-ray film. Phosphorylated proteins were analyzed first, then antibodies were removed using Western Blot Stripping Buffer (Thermo Fisher Scientific) before analyzing total protein. Membranes were stripped after imaging total proteins before analyzing loading control. DC9 cell line lysates were used as positive controls. Loading controls were assessed for each membrane and are presented with corresponding proteins analyzed on the same membrane.

Total, MHV68-specific, and dsDNA immunoglobulin levels were determined as previously described (49 (link)). Briefly, Nunc Maxisorp plates (Fisher Scientific, Pittsburg, PA) were coated with antigen of interest, either anti-IgG (heavy and light) or anti-IgM antibodies (Jackson ImmunoResearch, West Grove, PA), UV-irradiated MHV68 virus stock in PBS (740,000 microjoules/cm² × 2) (Stratalinker UV Crosslinker 1800; Agilent Technologies, Santa Clara, CA), or dsDNA from Escherichia coli (12.5 μg/ml; Sigma-Aldrich, St. Louis, MO) overnight at 4°C. Plates were washed with PBS containing Tween (PBS-Tween) (0.05%) and blocked for 1 h with PBS-Tween (0.05%)-BSA (3%), incubated with fivefold serial dilutions of serum in PBS-Tween (0.05%)-BSA (1.5%) for 2 h and then washed with PBS-Tween (0.05%). Bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse total IgG (heavy and light chain [H+L]) or IgM (Jackson ImmunoResearch, West Grove, PA) using 3,3′,5,5′-tetramethylbenzidine substrate (Life Technologies, Gaithersburg, MD). HRP enzymatic activity was stopped by the addition of 1 N HCl (Sigma-Aldrich, St. Louis, MO), and the absorbance was read at 450 nm on a model 1420 Victor³V Multilabel plate reader (PerkinElmer, Waltham, MA).