Supplementary Figure 2: KLHL6 WT, but not KLHL6 BTB-domain mutant, promotes roquin2 ubiquitylation and degradation. | Nature Cell Biology

Supplementary Figure 2: KLHL6 WT, but not KLHL6 BTB-domain mutant, promotes roquin2 ubiquitylation and degradation.

From: Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor roquin2

Supplementary Figure 2

a, Immunoblot analysis from immunoprecipitated endogenous KLHL6 in U2932 cells. IgG antibody immunoprecipitates = negative control. A representative blot from two independent experiments is shown. *Non-specific band. b, Immunoblot analysis from immunoprecipitated Flag–KLHL6 wild-type (WT) and mutants in HEK293T cells. EV, empty vector. c, Quantification of (x axis) and KLHL6 (y axis) protein levels in each DLBCL cell line. n = 11 DLBCL cell lines. r, Pearson correlation coefficient (95% confidence interval). d, Quantification of roquin1 and roquin2 immunoblots. Relative intensities were plotted over time for OCI-LY10 (left panel) and U2932 (right panel) (mean ± s.d., n = 3 independent experiments, two-way ANOVA, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s., not significant). e, Immunoblot analysis of whole-cell lysates from HEK293T (TET)-OFF cells transduced with retroviruses encoding a doxycycline (DOX) inducible expression of Flag-tagged KLHL6 carrying a hygromycin cassette (top panel). DOX was added and/or washed at the indicated times. Bottom panel shows the immunoblot analysis of the indicated proteins in HBL1 cells transduced with lentiviruses encoding a doxycycline (DOX) inducible expression of KLHL6 wild-type (WT) carrying a puromycin cassette. The cells were treated with DOX for 12 h. f, Immunoblot analysis of whole-cell lysates from OCI-LY8 cells transduced with retroviruses encoding empty vector (EV), KLHL6 wild-type (WT) or BTB-domain mutants (L65P, S94I and F97L) carrying a puromycin cassette. g, Immunoblot analysis of whole-cell lysates from HEK293T cells transduced with lentiviruses encoding a doxycline (DOX) inducible expression of KLHL6 carrying a puromycin cassette and infected with lentiviruses encoding empty vector(EV) or KLHL6 BTB-domain mutants (L65P and S94I) carrying a GFP marker. The cells were treated with DOX for 12 h. h, Immunoblot analysis of immunoprecipitated endogenous roquin2 in U2932 KLHL6+/+ and KLHL6−/− (clone-derived) cells treated with or without MG132 for 6 h. A representative blot from two independent experiments is shown. Unprocessed original scans of immunoblots for a,b,e–h are shown in Supplementary Fig. 8, and statistical source data for c,d and exact P values for d can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.

Back to article page