Journal: Frontiers in Oncology

Article Title: Proteolysis Targeting Chimeras for BTK Efficiently Inhibit B-Cell Receptor Signaling and Can Overcome Ibrutinib Resistance in CLL Cells

doi: 10.3389/fonc.2021.646971

Figure Lengend Snippet: BTK PROTAC leads to BCR signaling pathway inhibition. Peripheral blood CLL cells were cultured in 6-well dishes (20x10 6 cells/mL in RPMI 10% FCS) and incubated with 100 nM NC-1 for 18 hours or with ibrutinib (IBR) for 1 hour, at 37° in a humidified 5% CO2 atmosphere. DMSO treated cells served as controls. Following treatment, cells were incubated with goat F (ab’) 2 anti-human IgM (10 µg/mL) for 15 minutes or left unstimulated. Cells treated with ibrutinib were washed before activation. Then, proteins were extracted and analyzed by Western blot analysis. (A) A representative Western blot showing BTK (Tyr223), PLCγ2 (Tyr1217), Akt (S473) and ERK (T202/Y204) phosphorylation, as well as total amount of these proteins. Actin was used to verify equal loading. (B) Quantification of BTK, pBTK, pPLCγ2, pAkt and pERK levels in A by normalization to actin using myImageAnalysis™ Software (n=9). * p <0.05, ** p <0.01, *** p <0.001. (C) A representative Western blot showing pLyn (Tyr396), pCD79a (Tyr182), pSyk (Tyr525/526) and pShip1 (Tyr1020) levels,as well as total amount of these proteins. Actin was used to verify equal loading. (D) Quantification of Lyn, pLyn, pCD79a, pSyk and pShip1 levels in C by normalization to actin using myImageAnalysis™ Software (n=8). * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: ERK1/2, Phospho-ERK1/2 (Thr202/Tyr204), Akt (pan), phospho-Akt (S473),PLC γ2, pPLC γ2(Tyr1217),BTK, Phospho-BTK (Tyr223),Lyn(5G2), cleaved PARP (Asp214), CD79a, phospho-CD79a (Tyr182), Syk,phospho-Syk (Tyr525/526), SHIP1 and phospho-SHIP1 (Tyr1020) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Inhibition, Cell Culture, Incubation, Activation Assay, Western Blot, Phospho-proteomics, Software

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

Article Title: Activation of human peripheral IgM+ B cells is transiently inhibited by BCR-independent aggregation of Fc gammaRIIB.

doi: 10.4049/jimmunol.181.8.5350

Figure Lengend Snippet: FIGURE 8. FcRIIB aggregation affects Akt phosphorylation on Ser473. IIA1.6-FcRIIB (A) and IIA1.6-FcRIIBY292G (B) cells were treated or not stimulated (NS) with AT10-biotin and mouse anti-biotin (MABiotin) for the indicated periods of time (top). The same experience was monitored with IIA1.6-FcRIIB and IIA1.6-FcRIIBY292G cells in HBSS in absence of Abs (bottom). Cells were lysed and Akt phosphorylation (pAkt (Ser473)) was analyzed by Western blot as described in Materials and Methods. Data are representative of three independent experiments. Relative intensity was expressed as the ratio of pAkt to total Akt. C and D, Histograms represent the mean relative intensity S.D. of Akt phosphorylation in IIA1.6-FcRIIB (C) and IIA1.6-FcRIIBY292G (D) cells at different time points after FcRIIB aggregation. Results are from three independent experiments. Statistical analyses were performed using unpaired Student’s t test. , p 0.05 vs nonstimulated cells.

Article Snippet: Rabbit polyclonal anti-human phospho-Fc RIIB (Tyr292), polyclonal anti-SHIP-1, polyclonal anti-phospho-Akt (Ser473), and polyclonal anti-phospho-SHIP-1 (Tyr1020) Abs were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Fc γ RIIB regulates autoantibody responses by limiting marginal zone B cell activation

doi: 10.1172/JCI157250

Figure Lengend Snippet: ( A ) Simplified schematic diagram of key signaling molecules downstream of the BCR and FcγRIIB. ( B – D ) Representative examples and summary of FO and MZ B cells from control mice; cells were stimulated with 3 μg/mL intact anti-IgM (αIgM) or equimolar concentrations (2 μg/mL) of Fab′2 anti-IgM for 10 minutes, followed by Phosphoflow analysis of the phosphorylation of signaling molecules. ( E ) SHIP1 phosphorylation was analyzed by capillary Western blotting. ( F and G ) Representative examples and summary of p-Erk expression in FO and MZ B cells from control and Fcgr2b -cKO mice; cells were stimulated with intact anti-IgM as described in B – D . ( H – J ) PBMCs from healthy donors were stimulated with 15 μg/mL intact anti-IgM or equimolar concentrations (10 μg/mL) of Fab′2 anti-IgM for 60 minutes, after which the phosphorylation of signaling molecules was analyzed by Phosphoflow. ( H ) Representative example of Syk and Erk phosphorylation in MZ-like B cells. ( I and J ) Comparison of intact versus Fab′2 anti-IgM in naive and MZ-like B cells. The median percentage of inhibition calculated per donor is indicated. ( K – M ) Calcium flux following 75 μg/mL intact anti-IgM or equimolar concentrations (50 μg/mL) of Fab′2 anti-IgM. Peak calcium flux and the AUC were calculated using FlowJo. ( K ) Representative examples of calcium flux in naive and MZ-like B cells. ( L and M ) Comparison of the peak and AUC of calcium flux following intact versus Fab′2 anti-IgM in naive and MZ-like B cells. inh, inhibition. Data are shown as the median, with each symbol representing an individual mouse ( n = 4–7 mice per group for B – D ; n = 8 mice per group for G ; n = 6 mice per group for H – J ; n = 5 mice per group for L and M ; data for each were pooled from 2–3 independent experiments). * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: SHIP1 was detected with rabbit anti–mouse SHIP1 antibody (Cell Signaling Technology, polyclonal rabbit; catalog 2728; diluted at 1:10), followed by antibody stripping and p-SHIP1 (Tyr1020) detection with rabbit anti–mouse p-SHIP1 antibody (Cell Signaling Technology, polyclonal rabbit, catalog 3941; diluted at 1:10).

Techniques: Control, Phospho-proteomics, Western Blot, Expressing, Comparison, Inhibition