Goat f ab 2 anti human igm

Cells were stimulated at 37 °C for 15 min with 10 μg/mL of goat F(ab′)2 anti–human IgM (Southern Biotech). Cells were coated on a poly-L-lysine matrix then fixed 20′ with PFA 4% at RT. Cells were permeabilized with PBS + 0.1% Triton X-100 10′ at RT. To block unspecific staining, samples were treated for 1 h with PBS + 5% BSA at RT before staining. The following primary antibody was used in PBS 5% BSA buffer: rabbit monoclonal α-NF-Kappa-B1 p105/p50 (D4P4D) (13,586, CST). Samples were incubated overnight at 4 °C. For immunofluorescence, the following secondary antibody was used: goat anti-rabbit IgG labelled with Alexa 568 (Thermo Fisher Scientific) 1 h at RT in the dark. Slides were counterstained after three washes of PBS with 0.3 μg/mL 4,6-diamidino-2-phenylindole (Sigma-Aldrich). Images including Z-stacks were acquired on a Leica SP5 with an objective with × 63 magnification. Nuclear localization of NF-κB1/p50 was quantified by ImageJ software.

The Ramos cells (~3 × 10⁶) were famished of serum for 1 h and treated with ginsenosides for 1 h, and then washed with phosphate-buffered saline (Welgene, Korea), followed by stimulation with 1 μg/mL of goat F(ab’)2 anti-human IgM (Southern Biotech, Birmingham, AL, USA) for 10 min on ice. The cells were then lysed in a radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) and the proteins were separated by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The proteins were detected with anti-BTK Y223 (Cell Signaling Technology, Danvers, MA, USA), anti-BTK (53/BTK, Cell Signaling Technology) and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. The protein signals were monitored by chemiluminescence using Enhanced ChemiLuminescence (ECL) western blotting detection reagent (EMD Millipore), and the scanned blots were quantified using a Multi-Image analyzer (Fujifilm, Japan). The phosphorylation % of each lane was determined using Multi-Gauge software (Fujifilm). The IC₅₀ value was measured using control α-IgM+ set at 100% and control α-IgM− set at 0%.

293T cells were obtained from American Type Culture Collection (CRL-3216) and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Thermo Fisher Scientific). Nalm6 human pre-B cells (RCB1933, RIKEN Bioresource Research Center) were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and 5 × 10⁻⁵ M 2-mercaptoethanol. FACS-sorted naive B and DN B cells were cultured as Nalm6 and supplied with TLR7 agonist R848 (1 μg/mL, Invivogen), BAFF (10 ng/mL, R&D Systems, Bio-Techne), IL-21 (10 ng/mL, R&D Systems, Bio-Techne), IL-2 (50 ng/mL, R&D Systems, Bio-Techne), IFN-γ (20 ng/mL, Peprotech), and goat F(ab′)₂ anti-human IgM (10 μg/mL, SouthernBiotech). Four days later, the supernatants were collected, and the cells were further cultured in fresh media containing R848 and cytokines but without F(ab′)₂ anti-IgM. After 4 additional days, the culture supernatants were collected, and the quantity of IgG Abs was analyzed by ELISA.

Human E-selectin/Fc, human ICAM-1/Fc, human VCAM-1/Fc and human CXCL12 were from R&D Systems (R&D Systems, Minneapolis, MN, USA); goat anti-human IgM was from SouthernBiotech (SouthernBiotech, Birmingham, AL, USA); fluorescein isothiocyanate (FITC) goat secondary antibody to mouse was from Sigma (Sigma-Aldrich, St. Louis, MO, USA); KIM127 mouse monoclonal antibody was from American Type Culture Collection (ATCC, Rockville, MD, USA); 327A mouse monoclonal antibody was kindly provided by Dr. Kristine Kikly (Eli Lilly and Co., Indianapolis, IN, USA); goat F(ab’)₂ anti-human IgM was from Southern Biotech (Southern Biotech, Birmingham, AL, USA); Tyrphostin and rabbit polyclonal anti-actin antibody were from Sigma; Ibrutinib was from Selleck Chemicals (Selleck Chemicals, Houston, TX, USA); PE mouse anti-BTK (pY223) antibody was from BD Biosciences (BD Biosciences, San Jose, CA, USA); rabbit monoclonal anti-JAK2 (D2E12) was from Cell Signaling Technology (Danvers, MA, USA); siRNAs (ON-TARGETplus SMARTpool) were from Thermo Fisher Scientific (Fremont, CA, USA).

The antibodies used in this work, were as follows: Anti-14-3-3ζ (1:2000), anti-Pan 14-3-3 (1:2000), anti-MEF-2D (1:1000), anti-RBM25 (1:1000), anti-Bcl-x (1:1000), anti-pERK (44/42) (1:1000) and anti-ERK (1:1000) were purchased from Santa Cruz Biotechnology; anti-actin (1:100,000) from Sigma Aldrich; anti-RXRXXpS/T (1:1000) and anti-pS660 PKC βII (1:1000) from cell signaling; anti-pS473-AKT (1:5000) from R&D, anti-Pan-AKT (1:4000) was purchased from Invitrogen and goat F(ab’)2 anti-Human IgM from Southern Biotech. Polyclonal rabbit antibody against BTK-SH3 domain has been described earlier [22 (link)].