Journal: Nature

Article Title: Fundamental Roles of Chromatin Loop Extrusion in Antibody Class Switching

doi: 10.1038/s41586-019-1723-0

Figure Lengend Snippet: a, (upper and middle panels) Three individual repeats of CSR-HTGTS-Seq experiments shown in that employ a 5’Sμ bait to assay αCD40 region /IL4/TGFβ-stimulated IαΔ and IαΔ-i3CBEs cells. Junctions are plotted at 2.5 kb bin size. (bottom panels) Zoom-in views of three repeats of data in showing junctions located in the AID-targeted ectopic S region between Cγ2a and Iε in assays of IαΔ-i3CBEc cells. Junctions are plotted at 115 bp bin size. Blue lines indicate deletional joining and red lines indicate inversional joining. b, Bar graph shows percentages of junctions located in indicated AID targeted regions from IαΔ and IαΔ-i3CBEs cells. Data represents mean ± s.d. from three biologically independent repeats. P values were calculated via an unpaired two-tailed t -test based on the three repeats. c, AID targeting motif frequency analysis of 2 kb ectopic S region targeting peak and in comparably sized region just upstream and downstream of the targeting peak. d, FACS analysis of IgG3 and IgG2b surface expression in IαΔ and IαΔ-i3CBEs cells stimulated with αCD40/IL4/TGFβ for 72 hrs (6 biologically independent repeats). Bar graph shows percentages of IgG3 and IgG2b production from IαΔ and IαΔ-i3CBEs cells. Data represents mean ± s.d. from six biologically independent repeats. P values were calculated via an unpaired two-tailed t -test.

Article Snippet: APC IgM antibody (17-5790-82; eBioscienceTM), PE IgA antibody (1100-09S; SouthernBiotech), PE IgG3 antibody (1100-09S; SouthernBiotech), and PE IgG2b antibody (1090-09S; SouthernBiotech) were used for flow cytometric analysis.

Techniques: Two Tailed Test, Expressing

Journal: Nature

Article Title: Fundamental Roles of Chromatin Loop Extrusion in Antibody Class Switching

doi: 10.1038/s41586-019-1723-0

Figure Lengend Snippet: a, Illustration of dominant, deletional CSR between Sμ and Sα in CH12F3 cells. b, Illustration of Cas9/gRNA targeting (lightning bolts) used to generate the CH12F3NCΔ line. c, Southern blot confirmation (using BamHI digestion and a J H 4 probe) of the CH12F3NCΔ lines (done twice independently with similar results). d, Western blot confirmation of AID expression or lack of expression, respectively, in AID sufficient and deficient (via targeted deletion) CH12F3NCΔ and IαΔ lines following stimulation with αCD40/IL4/TGFβ for 72 hrs (done twice independently with similar results). e, FACS analysis for surface IgA expression in CH12F3NCΔ-AID −/− cells stimulated with αCD40/IL4/TGFβ for 72 hrs (done three times independently with similar results). f, Three repeats of CSR-HTGTS-Seq data shown in for αCD40/IL4/TGFβ-stimulated CH12F3NCΔ and IαΔ cells (3 biologically independent repeats). Junctions are plotted at 2.5 kb bin size. The blue lines indicate deletional joining and the red lines indicate inversional joining. Bar graph shows percentages of junctions located in different regions from CH12F3NCΔ and IαΔ cells. Data represents mean ± s.d. from three biologically independent repeats. P values were calculated via an unpaired two-tailed t -test. g, FACS analysis of IgA, IgG3 and IgG2b surface expression in CH12F3NCΔ and IαΔ cells stimulated with αCD40/IL4/TGFβ for 72 hrs (4 biologically independent repeats). Bar graph shows percentages of IgA, IgG3 and IgG2b expression on CH12F3NCΔ and IαΔ cells. Data represents mean ± s.d. from four biologically independent repeats. P values were calculated via an unpaired two-tailed t -test.

Article Snippet: APC IgM antibody (17-5790-82; eBioscienceTM), PE IgA antibody (1100-09S; SouthernBiotech), PE IgG3 antibody (1100-09S; SouthernBiotech), and PE IgG2b antibody (1090-09S; SouthernBiotech) were used for flow cytometric analysis.

Techniques: Southern Blot, Western Blot, Expressing, Two Tailed Test