Mouse anti smad2

OVCAR-3 and SK-OV-3 cells were lysed in RIPA lysis buffer (Thermo Scientific, Rockford, USA) with 1 % PMSF (Beyotime) and 1 % phosphatase inhibitor (KeyGEN), followed by sonication. The cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. Equal amount proteins were separated on 15 % SDS-PAGE with 4X Protein SDS PAGE Loading Buffer (TaKaRa) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5 % non-fat milk in Tris-buffered saline with Tween 20 (TBS-T) for 1 h, the membrane was incubated with a primary antibody at 4 °C overnight and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:10,000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. The following primary antibodies were used: rabbit anti-B2M (1:3000 dilution, Abcam), mouse anti-Smad2 and rabbit anti-phospho-Smad2 (both 1:2000 dilution, Cat# 3103 and #3101, Cell Signaling Technology, Inc.) and rabbit anti-β-actin (1:5000 dilution, Cat# 66009-1-Ig, Proteintech, Wuhan, China). Signals were detected using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) and quantified using Tanon-4500 Gel Imaging System with GIS ID Analysis Software v4.1.5 (Tanon Science and Technology Co., Ltd., Shanghai, China).

OVCAR-3 and SK-OV-3 cells were lysed in SDS lysis buffer (Beyotime, Haimen, China) with 1% PMSF (Beyotime), followed by sonication. Equal amount of protein was separated on 15% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBS-T) for 1 h, the membrane was incubated with a primary antibody at 4°C overnight and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:3,000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. The following primary antibodies were used: rabbit anti-CSTB (1:10,000 dilution, Abcam), mouse anti-Smad 2 (1:2,000 dilution), rabbit anti-phospho-Smad2 (1:2,000 dilution), and rabbit anti-β-actin (1:5,000 dilution) (Cell Signaling Technology, Inc.). Signals were detected using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) and quantified using Tanon-4500 Gel Imaging System with GIS ID Analysis Software v4.1.5 (Tanon Science & Technology Co., Ltd., Shanghai, China).

Cellular lysates were prepared using RIPA buffer. Cellular proteins (25 mg) were resolved by polyacrylamide-SDS gel electrophoresis and transferred to polyvinylidene difluorid membranes (Millipore) using a standard procedure. The membranes were incubated with mouse anti-P-Smad2 (1:1000, Abcam), mouse anti-Smad2 (1:1000, Abcam), mouse anti-P-Smad3 (1:1500, ProteinTech), mouse anti-Smad3 (1:1500, ProteinTech), rabbit anti-E-cadherin (1:1000, Abcam), rabbit anti-vimentin (1:1000, Abcam), and rabbit anti-PAI-1 (1:1000, Abcam) antibodies. Anti-rabbit or anti-mouse IgG was used as secondary antibody (1:8000). Densitometry was done using Quantity One 4.4.0 software.