Journal: Cell

Article Title: LAP2 Proteins Chaperone GLI1 Movement Between Lamina and Chromatin to Regulate Transcription

doi: 10.1016/j.cell.2018.10.054

Figure Lengend Snippet: (A) BASU-GLI1 vicinal labeling in ASZ followed by streptavidin pulldown ± CRT0329868 (CRT) (+biotin, +CRT, 5hr). (B) APEX2-GLI1 vicinal labeling in ASZ followed by streptavidin pulldown ± PSI. (C) Co-IP of FLAG-GLI1 in ASZ ± PSI followed by immunoblot. (D) PLA between total GLI1 and LAP2α (top) or LAP2β (bottom) in ASZ treated with indicated inhibitors for 2hr (scale bar=20μm, n=10 fields, ANOVA). (E) PLA between total GLI1 and LAP2α (left) or LAP2β (right) in 1º human BCCs treated with vorinostat ex vivo (scale bar= 66μm, n=10 fields, ANOVA). (F) Co-IP of in vitro translated HA-GLI1 zinc-finger domain (HA-GLI1ZF) from WCE. Inputs in Figure S5B. (G) LAP2-binding mutants mapped onto GLI1:DNA crystal structure (pdb:2GLI). Mutations which inhibit (red) or are permissive of (grey) LAP2 binding are illustrated as spheres. Co-IP in Figure S5C. (H) Co-IP of HA-GLI1WT/T296E transfected into HEK293T followed by immunoblot of endogenous LAP2. Inputs in Figure S5D. (I) qRT-PCR of GLI1 and GAPDH following transfection of GLI1WT/T296E into NIH3T3 (n=9, ANOVA). Associated immunoblot in Figure S5E. (J) Co-IP of full length GLI1 (GLI1 FL) or zinc-finger domain GLI1 (GLI1 ZF) with recombinant LAP2 constant region (−/+ indicate the addition of LAP2 peptide). Input in Figure S5F. (K) Co-IP of wheat germ cell extract in vitro translated HA-GLI1 with chemically synthesized biotin-LEM-like (residues 5–48), biotin-LEM (residues 109–153), or biotin-scrambled LEM-like domains. Associated inputs and saturation binding experiment in Figure S5G and S5H. (L) Co-IP of FLAG-GLI1 co-transfected into HEK293T with a gradient of LAP2α, followed by immunoblot for total LAP2 (n=3). Input and reciprocal IP in Figures S5I and S5J. (M) GLI1 transfected in HEK293T (top, cellular IP) or in vitro translated and incubated in WCE (bottom three, in vitro IP) with indicated mutations/truncations co-IP with associated epitope tag. IP washed over a gradient of high salt conditions prior to immunoblot. Complex strength=−slope(x)−1−slope(α)−1 Error bars represent standard error, error bars omitted when smaller than the width of associated data point symbol, ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S5.

Article Snippet: Curve fitting for FRAP analysis and saturation binding was also performed in GraphPad PRISM 6.

Techniques: Labeling, Co-Immunoprecipitation Assay, Western Blot, Ex Vivo, In Vitro, Binding Assay, Transfection, Quantitative RT-PCR, Recombinant, Synthesized, Incubation