Figure 3

MCL-1 protein is stabilized in GC B cells. (a) Ratios of protein (average of quantified western blot experiments shown in Figure 2b, with n=2–4) over mRNA (average of real time qPCR experiments shown in Figure 1d, with n=3–5) in B-cell subsets for MCL-1, BCL-2 and BCL-XL expressed in arbitrary units (a.u.) (b) Western blot analysis on FACS-purified naive or GC B cells for MCL-1 and Actin after culture for 0, 2 or 4 h (h) with cycloheximide (CHX), an inhibitor of protein synthesis. Data are representative of two individual experiments. (c) Real time qPCR of known MCL-1-specific ubiquitin ligases Mule (HUWE1), FBW7 (FBXW7) and βTrCP1 (BTRC) in purified cells as shown in Figure 1a, corrected for expression of household gene Hprt and relative to naive B cells. Data are average of five experiments with S.E.M. Statistics were calculated in relation to Bn cells. (d) Western blot analysis on MACS-enriched GC B cells for MCL-1 and Actin after culture for 0, 2 or 4 h (h) with cycloheximide (CHX), with or without PP2A-inhibitor okadaic acid (OA). Cells in (b) and (d) were cultured in standard tissue culture medium (Iscove's modified Dulbecco's media) supplemented with 10% (v/v) heat-inactivated fetal calf serum and antibiotics, but without additional cytokines. *P⩽0.05, **P⩽0.01