Differential downstream p65 nuclear translocation in TACI isoform–transduced B-cell signals through MyD88 and CAML. (A) Confocal microscopy of nuclei isolated from A20 nontranduced cells, A20 GFP, A20 with TACI short or long isoforms, and nonsignaling TACI S194X mutant cells, cultured as in Figure 3, in the presence or absence of 100 ng/mL of rhBAFF or rhAPRIL, and stained for p65 (red); nuclei were counterstained with DAPI (blue). (B) Quantitative assessment of p65 nuclear translocation (relative to cell number), represented as p65 relative intensity. Error bars represent standard error of the mean. *P < .05; **P < .01; 2-tailed unpaired Student t test. (C) Immunoblot analysis of p65 nuclear translocation after subcellular fractionation of resting cells after 6-hour starvation (C: cytoplasmic fraction; N: nuclear fraction; GAPDH: cytoplasmic fraction loading and purification control; and RNApol II: nuclear fraction loading and purification control). For quantitation of nuclear p65 levels, >200 cells were microscopically assessed and analyzed with ImageJ software. (D) In resting transduced A20 cells, colocalization of TACI isoforms with MyD88 and CAML was observed by confocal microscopy, as marked by arrowheads. Staining: TACI (green), MyD88 (red) or CAML (red), along with DAPI (blue) to stain nuclei. Residual colocalization of S194X mutant TACI with CAML was also observed. The mean percentage and standard error were calculated from 3 independent experiments. Images in panels A and D show white dividing lines for cells taken from the same field; original magnification ×63. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RNApol II, RNA polymerase II.