Fig. 3: LATS1 interacts with and stabilizes Beclin-1 in a kinase activity-independent manner.
From: LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function
a HEK293T/17 cells were transfected with indicated plasmids. Seventy-two hours later, cell lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with indicated antibodies. Input represents lysates directly immunoblotted without immunoprecipitation. Results represent three independent experiments. b Huh7 cells were lysed for immunoprecipitation with indicated antibodies followed by immunoblotting analysis for Beclin-1 and LATS1. Input represents lysates before immunoprecipitation. Results represent three independent experiments. c HEK293T/17 cells transfected with indicated plasmids were treated with sorafenib (SRF; 10 μM, 2 h) followed by immunoprecipitation with indicated antibody. Results represent three independent experiments. d Huh7p or IR or CR cells were lysed for immunoprecipitation with indicated antibodies followed by immunoblotting analysis for Beclin-1 and LATS1. Input represents lysates before immunoprecipitation. Results represent three independent experiments. e LATS1 promotes Beclin-1 stability in a dosage-dependent manner. HEK293T/17 cells were transfected with same amount of vector encoding for Flag-tagged Beclin-1 together with increased amounts of vectors encoding for Myc-tagged wild-type LATS1 or kinase-dead mutant LATS1 (KD D846A) as indicated. Seventy-two hours later, cells were lysed and analyzed by immunoblotting for the expression of Beclin-1 (Flag) and LATS1 (Myc). Results represent three independent experiments. f, g HEK293T/17 cells transfected with indicated plasmids were treated with cycloheximide (CHX; 40 μg/mL) for the indicated times. Representative Immunoblots was shown in f and quantification g of band intensity was pooled from three independent experiments. Statistical significance was done using one-way ANOVA. **P < 0.01. Note: to achieve equal amount of Beclin-1 protein level at time point 0, amounts of Flag-Beclin-1 plasmid transfected between empty vector and LATS1 were 3:1. h Huh7 cells transfected with indicated siRNAs were treated with DMSO or sorafenib (Srf; 10 μM) for 48 h. Cell lysates were analyzed by immunoblotting with indicated antibodies. A representative immunoblot from three independent experiments is shown. i Liver tissues from Control mice (Lats1fl/fl; Control; n = 3) or mice with a hepatocyte-specific depletion of Lats1 (Alb-Cre;Lats1fl/fl; cKO; n = 3) were collected at young adult age (12–16 weeks) or at old age (55–65 weeks) and lysed for immunoblotting analysis.
