Extended Data Fig. 12: Targeting non-GFP tagged proteins with novel effectors.
From: Proteome-scale discovery of protein degradation and stabilization effectors
(a) HeLa cells were co-transfected with 3xFLAG-V5-KRAS and indicated effectors fused to an intracellular single domain antibody (iDab) targeting Ras or LMO2 (control). (b) 293T cells were co-transfected with IKBKE fused to ALFA-3xFLAG tag and indicated effectors fused to Nb(ALFA)-Myc, followed by western blotting for IKBKE (anti-FLAG), effector (anti-Myc), and Hsp90. (c) Indicated effectors fused to Nb(ALFA)-Myc were co-transfected with ALFA-3xFLAG-ARAF into 293T cells, followed by western blotting for ARAF (anti-FLAG), effector (anti-Myc), and Hsp90. (d) Stable HCT116 cell lines expressing doxycycline-inducible effectors fused to WDR5-targeting monobody Mb(WDR5) were treated with 1 µg/ml doxycycline for 48 h or left untreated. Top, Endogenous WDR5 levels and effector expression were assessed by western blotting. Bottom, Quantification of WDR5 levels after doxycycline induction. Statistical significance was calculated with an unpaired t-test with false discovery rate correction for multiple hypotheses. (e) Correlation between K562 cell proliferation and BCR-ABL levels in cells stably expressing effectors fused to Mb(ABL) treated with doxycycline for 6 days. (f) Volcano plots of the p-value versus the log2 fold change of proteins in K562 cells transduced with indicated doxycycline-inducible effector fused to Mb(ABL)-HA treated with doxycycline for 24 h versus control. (g) Correlation between FBXL15 and FBXL12 proteomics profiles in K562. (f and g) Each dot represents a protein. Points colored black, blue, and orange indicate the endogenous BCR-ABL fusion protein, FBXL12-Mb(ABL)-HA fusion protein, and FBXL15-Mb(ABL)-HA fusion protein, respectively. At least two independent experiments were performed for (a), (b), (c) and (d). Results from one representative experiment are shown. (h) Mouse xenograft model of K562 chronic myeloid leukemia cells expressing effectors fused to BCR-ABL targeting Mb(ABL) monobody. Doxycycline inducible K562 cell lines expressing the listed effectors were subcutaneously injected into NSG mice (10 mice per cell-line). 2 weeks post transplantation, mice were randomly divided into two groups: vehicle (no Dox) and doxycycline (+ Dox). Tumor growth was measured with a caliper. Data are presented as mean values +/− SD. N = 5 animals *, p≤0.0389; **, p≤0.0074; ***, p≤0.0006; ****, p≤0.0001 (two-way ANOVA with Geisser-Greenhouse correction and Šídák correction for multiple comparisons).
