Supplementary Figure 4: The roquin2(Y691F) mutant is resistant to KLHL6-mediated degradation.
a, Schematic representation of the sequence of the biotinylated roquin2 peptides. b, A pull-down assay using the indicated amount of biotinylated roquin2 peptides incubated with cell extracts from HEK293T cells stably expressing KLHL6. AP, Affinity purification. c,d, Same as in b except that Flag-tagged in vitro translated proteins as indicated were used instead of cell extracts. Immunoblot analysis for the indicated proteins was performed using Flag antibody. e, Quantification of immunoblots from BJAB cells stably expressing roquin2(WT) or roquin2(Y691F). Relative intensities were plotted over time under cycloheximide (CHX) treatment (mean ± s.d., n = 3 independent experiments, two-way ANOVA, *P ≤ 0.05; **P ≤ 0.01). f, Immunoblot analysis of whole-cell lysates from HEK2932T cells stably expressing KLHL6(WT) or KLHL6(L65P) carrying hygromycin cassette and further infected with retroviruses encoding an empty vector (EV), roquin2(WT) or roquin2(Y691F) carrying a puromycin cassette. A low exposure (l.e.) and high exposure (h.e.) are shown. g, Immunoblot analysis for indicated amounts of recombinant roquin2 (set as the standard) along with whole-cell lysates from U2932 stably expressing HA–roquin2(WT) and HA–roquin2(Y691F) (left panel) or U2932 KLHL6+/+ and KLHL6−/− cells (clone-derived) (middle panel). Right panel shows intensity of quantified roquin2 bands compared to the standard. A representative blot from one experiment is shown. h, Flow cytometry analysis of GFP+-live OCI-LY10 KLHL6+/+ and KLHL6−/− (clone-derived) cells infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker. Cells were grown in media containing 2 μg ml−1 of F(ab′)2–IgM and normalized to the shCTRL cells set as 100% (mean ± s.d., n = 3 independent experiments, two-way ANOVA, **P ≤ 0.01; ****P ≤ 0.0001). i, Immunoblot analysis of whole-cell lysates from GFP-sorted U2932 (left panel) and OCI-LY10 (right panel) KLHL6+/+ and KLHL6−/− (clone-derived) infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker. A representative blot from two independent experiments is shown. Unprocessed original scans of immunoblots for b–d,f,g,i are shown in Supplementary Fig. 8, and source data for g and statistical source data and exact P values for e,h can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.
