Ab70561

HGC-27 and MKN-28 cells were harvested and extracted using lysis buffer (100 mM Tris-HCl, 2% SDS, 1 mM Mercaptoethanol, 25% Glycerol). Cell extracts were boiled in loading buffer and equal amount of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride (PVDF) membranes. The primary antibodies-anti-LATS1 (ab70561, Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA), anti-YAP (ab52771, Rabbit monoclonal antibody, Abcam, Cambridge, MA, USA), anti-p-YAP (S127) (ab76252, Rabbit monoclonal antibody, Abcam, Cambridge, MA, USA) and anti-GAPDH (ab153802, Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA) were diluted at a ratio of 1:1000 according to the instructions and incubated overnight at 4 °C. Horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:10,000, and incubated at room temperature for 1 h. The membranes were washed with PBS for three times and the immunoreactive bands were visualized using ECL-PLUS/Kit (GE Healthcare, Piscataway, NJ, USA) according to the kit’s instruction.

Antibodies: YAP (ab56701, abcam, 1:5000), Lats1 (ab70561, abcam,1:5000), p-YAP (ab62751, abcam,1:1000), Bax (ab32503, abcam,1:5000), Bcl-2 (ab196495, abcam,1:1000), TAZ (ab224239, abcam,1:5000), Caspase-3 (ab13847, abcam,1:500), GAPDH (ImmunoWay, YM3029,1:5000), Mst1 (CST,3682s,1:1000), TEAD1 (ab133533, abcam,1:5000), Cleaved-caspase3 (CST,9664s,1:1000), Phospho-Lats1/Lats2 (PA5-64591,Invitrogen,1:1000), Histone H3 (ImmunoWay, YM3038,1:5000).
Briefly, protein concentrations of tissues or cells were quantified by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Sample of proteins (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to PVDF membrane, then blocked by 5% non-fat milk in PBST buffer (PBS buffer containing 0.05% Triton-100) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4°C, after 3 times washing in PBST buffer, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Signals were detected by using an ECL substrate (Thermo Scientific) and exposure with the Tanon 5500 imaging system. The intensity of the bands was quantified by densitometry.

Total cell protein was extracted from BMSCs according to the SAB Total Protein Extraction Kit (SAB, USA) kit instructions from BMSCs. A BCA (Beyotime, China) assay kit was used to measure the protein concentration. 30 micrograms of protein from each sample was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with skim milk, the membranes were incubated with the following primary antibodies overnight at 4℃: Bmal1 (ABCAm, ab93806), GAPDH (CST, 2118 S), YAP (ABCAm, ab205270), p-YAP (ABCAm, ab76252), TAZ (ABCAm, ab84927), MST1 (CST, 3682 S), MST2 (CST, 3952 S), P-MST1/2 (CST, 49,332 S), LATS1 (ABCAm, ab70561), LATS2 (ABCAm, ab135794), ALP (Affinity, DF12525), RUNX2 (Bioss, bs-1134R), OCN (Affinity, DF12303), P53 (ABCAm, ab26), P16 (ABCAm, ab80) and β-actin (Abmart, P30002). The antibodies were diluted in Tris-buffered saline (TBS) with Tween-20 (TBST) at 1:1000. Subsequently, the membranes were immersed in HRP universal secondary antibody (1:2000, CST, 7074,) for 1 h. Afterwards, the protein bands were visualized according to the ECL kit (Millipore, USA), and the gray values were analyzed using ImageJ software.