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. Author manuscript; available in PMC: 2019 Jul 11.
Published in final edited form as: Oncogene. 2019 Jan 11;38(17):3316–3324. doi: 10.1038/s41388-018-0668-3

Fig. 2.

Fig. 2

BTK is activated in PanIN-associated regulatory B cells. a Representative immunofluorescent staining of 2 month old KC mice (N=6) (31) pancreata to detect co-localisation of pBTK (Y223) with IL-10 and B cell marker CD19. Mice pancreata were fixed, processed and stained as described previously (7). Antibodies used were pBTK (Y223) (1:200, 109336, LSBio, Seattle, WA, USA), CD19 (1:50, 550284, BD Biosciences, San Jose, CA, USA) and IL-10 (1:50, sc-365858, Santa Cruz Biotechnology, Dallas, TX, USA). Slides were examined and imaged on a Nikon Eclipse Ti2 microscope. Scale bar, 100 μm. b Representative intracellular flow cytometric analysis of pBTK (Y223) in B cells from pancreata of KC mice (31), as described previously (7). Single cell suspension from pancreatic tissue was prepared as described previously (7) and stained with the following antibodies for flow cytometry analysis: CD45 (2D1, 368521, Biolegend); CD19 (6D5, #115506, Biolegend), CD1d (1B1, 123509, Biolegend), CD5 (53–7.3, 100621, Biolegend), pBTK (Y223) (N35–86, 564848, BD). After gating on the CD45+ and CD19+ population, cells were analysed for pBTK (Y223) expression in CD1dhiCD5+ Bregs. Also shown are isotype controls for pBTK (Y223) staining. Graph represents quantification of FACS analysis, indicating percentage of CD1dhiCD5+ Breg cells expressing pBTK (Y223) in KC mice (2–3 mo old, N=5). Error bars indicate SD. P-value calculated using Student t test (two tailed, unpaired). **, P≤0.01. c Experimental schematic for pBTK analysis of KrasG12D-PDEC orthotopic lesions in wild type or IL1R1−/− mice. d Western blot analysis of pBTK expression in PanIN-associated wild type or IL1R1−/− B cells. Total B cells were isolated from 2 week old pancreatic orthotopic lesions implanted in wild type (C57BL/6J, Jackson Laboratory, Bar Harbor, ME, USA) or IL1R1−/− mice (B6.129S7-Il1r1tm1Imx/J, Jackson Laboratory) (N=5) using CD45R (B220)-linked MACS beads (130–049-501, Miltenyi) and MS columns (130–042-201, Miltenyi) as per manufacturer instructions. Isolated cells were lysed in sample buffer and analysed by immunoblotting on a LiCor Odyssey imager using the following antibodies: Phospho-BTK (Tyr 223) (5082S, Cell Signaling Technologies, Danvers, MA) and BTK (8547S, Cell Signaling Technologies).