Journal: PLOS One
Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions
doi: 10.1371/journal.pone.0342107
Figure Lengend Snippet: (A-B) LIMD1-KO MCF10A cells stably expressing GFP tagged wild-type LIMD1 (WT) or LIMD1 strain insensitive mutants (F512A, F575A, and Y646A, see also .) were established by lentiviral transduction and imaged using fixed and live-cell imaging. (A) The indicated cell lines were stained using anti-LATS1 and anti-TRIP6 antibodies. Merged images show LATS1 (red), TRIP6 (green) and DNA (blue). (B) The indicated cell lines from part (A) were imaged live for GFP fluorescence. (C) Quantification of the LATS1 junction to cytoplasm mean intensity ratio. Error bars represent the standard deviation. ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the plot (mean ± SD; n = 5; ****P < 0.0001). (D-E) LATS1/2 were tested for their ability to bind to strain insensitive mutants of LIMD1 by co-immunoprecipitation. (D) LATS2 was tested for its ability to bind to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1 and LATS2-FLAG were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged WT and mutants of LIMD1 were combined with LATS2-FLAG lysates. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (V5) and LATS2-FLAG (LATS2). (E) LATS1 was tested for binding to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1, and LATS1-3xMyc were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged LIMD1 variants were combined with LATS1-3xMyc lysates. Anti-Myc or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (LIMD1) and LATS1-3xMyc (LATS1). (F) Quantification of relative amounts of LATS2 (normalized to wild-type LIMD1 in the IP fraction) immunoprecipitated by wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants in part (D) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; ***P = 0.001, ****P < 0.0001). (G) Quantification of relative amounts of wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants (normalized to wild-type LIMD1 in the input fraction) immunoprecipitated by LATS1-3xMyc from part (E) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; *P < 0.05, ***P = 0.001).
Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.
Techniques: Stable Transfection, Expressing, Transduction, Live Cell Imaging, Staining, Fluorescence, Standard Deviation, Immunoprecipitation, Transfection, Control, Western Blot, Binding Assay
Bethyl is a verified supplier 
