Rabbit anti phospho lats1

Western blot was done by running 10% SDS-PAGE, following standard blotting protocol. Primary antibodies used in this study include: rabbit anti-cleaved Caspase 3 (1:1000, Cell Signaling), rabbit anti-YAP (1:1000, Santa Cruz), rabbit anti-Phospho-YAP (1:500, Cell Signaling), rabbit anti-Lats1 (1:500, Cell Signaling), rabbit anti-Phospho-Lats1 (1:500, Cell Signaling), rabbit anti-Phospho-Mst1/2 (1:500, Cell Signaling), and mouse anti-α-Tubulin (1:2000, Sigma) antibodies. Secondary antibodies are donkey anti-rabbit or mouse IgG antibodies (Amersham).

Brain tissues were dissected from Htt-KI mice or littermate control mice and washed three times with ice-cold PBS and dissolved in lysis buffer containing 62.5 mM Tris–HCl (pH 8.0), 2% (w/v) SDS, 2.5% (v/v) 2-mercaptoethanol and 5% (v/v) glycerol. The protein concentration was quantified using the BCA method (Micro BCA Protein Assay Reagent Kit, Thermo Fisher Scientific, MA, USA). Primary and secondary antibodies were diluted for immunoblotting as follows: rabbit anti-LATS1 (1:2000, Cell Signaling Technology, MA, USA, #3477), rabbit anti-phospho-LATS1 (Ser909, 1:5000, Cell Signaling Technology, MA, USA, #9157), mouse anti-PLK1 (1:2000, Invitrogen, MA, USA, #37-7000), anti-phospho-PLK1 (Thr210, 1:30000, Abcam, Cambridge, UK, #ab155095); HRP-conjugated anti-mouse IgG (NA931VA) and anti-rabbit IgG (NA934VS) (both of them, 1:3000, GE Healthcare, Buckinghamshire, UK). Antibodies were diluted in Can Get Signal (TOYOBO, Osaka, Japan). ECL prime (GE Healthcare, Buckinghamshire, UK) was used to detect the bands using LAS4000 (GE Healthcare, Buckinghamshire, UK) [8 ].

Immunohistochemistry was performed as previously described with minor modifications [8 ]. After deparaffinization, rehydration, and antigen retrieval (microwaved in 10 mM citrate buffer, pH 6.0, at 100 °C, 5 min, three times), the sections were incubated sequentially with 0.5% TritonX-100 in PBS for 30 min at room temperature (RT) to membrane permeation, with 10% FBS for 60 min at RT, with primary antibodies: rabbit anti-phospho-LATS1 (Ser909, 1:100, Cell Signaling Technology, MA, USA, #9157), rabbit anti-phospho-PLK1 (Thr210, 1:100, Abcam, Cambridge, UK, #ab155095), mouse anti-MAP2 (1:200, Santa Cruz, TX, USA, #sc-32791) and mouse anti-NeuN (1:100, Abcam, Cambridge, UK, ab104224) one or two overnight, and finally with secondary antibodies: Alexa Flour 488-labeled anti-mouse IgG (1:1000, Invitrogen, MA, USA) and Cy3-labeled anti-rabbit IgG (1:500, Jackson ImmunoResearch, PA, USA) for 1 h at RT. Images were acquired by confocal microscopy: Olympus FV1200 (Olympus, Tokyo, Japan) and LSM710 (Carl Zeiss, Oberkochen, Germany).