Journal: Frontiers in Immunology
Article Title: CK2β-regulated signaling controls B cell differentiation and function
doi: 10.3389/fimmu.2022.959138
Figure Lengend Snippet: Lack of CK2β alters the activation and expression of molecules important for the GC reaction. (A) WB of pERK1/2 and ERK1/2 in purified splenic B cells at d=0 and d=3 of LPS and LPS+IL4 stimulation. This figure is representative of three independent experiments. (B, C) WB analysis of purified splenic B cells unstimulated and after anti-CD40 ± IL-4. (B) Stimulation for 5 and 10 minutes and (C) for 48 hours. Blots are representative of four independent experiments. In (A-C) at least two spleens were pooled together in each experiment. (D, E) Evaluation by qRT-PCR of Aicda levels in purified splenic B cells after stimulation with LPS ± IL-4 (D) or anti-CD40 ± IL-4 (E) . The expression is corrected for Gapdh levels and normalized to CK2β CTRL B cells. Data are shown as mean ± SD (CK2β CTRL n=3; CK2β κO n=3). Statistical significance is determined by Student’s t test (*p < 0.05). (F) Analysis of spleen sections from CK2β CTRL and CK2β κO mice after immunization with SRBC (14 days) stained with RNAscope probes to detect Aicda levels in GCs and counterstained with hematoxylin. In order from top to bottom 4X, 10X, 20X objective magnifications. Images are representative of three controls and three CK2β κO mice. (G) Quantitative analyses of Aicda in situ hybridization signals upon immunization with SRBC for 14 days. Graphs represent mean ± SD obtained by calculating the average percentage of positive signals in five non-overlapping fields for each mouse at high-power magnification using the Positive Pixel Count v9 ImageScope software, Leica Biosystems (n= 3 CK2β CTRL and 3 CK2β κO mice). Statistical significance is determined by Nested t test (*p < 0.05). (H) Bcl6 , Irf4 and Prdm1 levels determined by qRT-PCR on purified B cells treated for 48h with anti-CD40 ± IL-4. Data were normalized over Gapdh and CTRL samples, and represented as mean ± SD (n=3). Statistical significance was determined by Student’s t test (*p < 0.05; **p < 0.01). In (A–E, H) the B cell fraction was purified with EasySep™ Mouse B Cell Isolation Kit (Stemcell) and purity was >97%.
Article Snippet: AICDA transcript (ID: 11628) was detected using RNAscope 2·5 HD Detection Reagent-BROWN (Advanced Cell Diagnostic) in accordance with the manufacturer’s protocol.
Techniques: Activation Assay, Expressing, Purification, Quantitative RT-PCR, Staining, RNAscope, In Situ Hybridization, Software, Cell Isolation
