Rabbit anti vimentin

The immunofluorescence staining was performed on cells grown on 22 × 22 mm coverslips. OVCAR-3 cells (3 × 10⁴/well) were grown in a 12-well plate and cultured in the 37°C incubator overnight. At 72 h after the transfection with 80 nM siRNA-FOXA1, 4% w/v paraformaldehyde was used for fixation for 30 min. 0.5% v/v Triton X-100 was used in permeabilization for 5 min and 10% normal donkey serum for blocking for 1 h at room temperature. Then, the cells were incubated at 4°C overnight with the primary antibodies: mouse anti-E-cadherin (1 : 500, Abcam, USA) and rabbit anti-vimentin (1 : 500, Abcam, USA). After being washed by PBS for 3 times, secondary antibodies listed as follows were used for incubation for 1 h: Alexa Fluor® 488 donkey anti-mouse IgG or anti-rabbit (1 : 200, Jackson ImmunoResearch, USA). The cells were finally counterstained with DAPI (Beyotime, China) for 5 min, and the coverslips were mounted by using 10 μL of FluroGuard antifade solution (Bio-Rad, USA). Images were taken using a confocal microscope (Leica, Germany).

Briefly, total protein was isolated from ground frozen atrium tissues and CFs using RIPA lysis buffer (Beyotime, China). The samples were clarified by centrifugation at 13 800 g for 15 minutes at 4°C. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, China). A total of 20 μg of protein was separated on 10% SDS‐PAGE at 80‐100 V for 2 hours and transferred onto PVDF membranes (Millipore, USA) at 300 mA for 1.5 hours. The membranes were blocked with 5% non‐fat milk in Tris‐buffered saline with tween 20 (TBST) for 1 hour at room temperature. Each membrane was incubated with primary antibodies at 4°C overnight and then secondary antibodies at room temperature for 1 hour the next day. The following antibodies were used: rabbit anti‐TGFβRII (1:500, Santa Cruz), rabbit anti‐α‐SMA (1:1000, Abcam), rabbit anti‐Col I (1:1000, Abcam), rabbit anti‐vimentin (1:1000, Abcam), mouse anti‐Col3α1 (1:250, Santa Cruz) and mouse anti‐GAPDH (1:2000, Abcam) antibodies. All proteins were visualized using the ECL reagent Kit (Millipore Corp, USA). The values of the band intensities were quantified using Image J software.

Briefly, cells were washed with phosphate buffered saline (PBS) before harvesting with lysis buffer (Sangon Biotech, Co., Ltd., Shanghai, China). Protein was separated by SDS‐PAGE and transferred to a Polyvinylidene Fluoride (PVDF) membrane. Non‐specific binding was blocked with Tris Buffered Saline Tween (TBST) containing 5% (w/v) non‐fat dried milk. The membrane was then incubated with rabbit anti‐ATF3 (1:1000; Abcam, Cambridge, MA, USA), rabbit anti‐α‐catenin (1:50000; Abcam), rabbit anti‐E‐cadherin (1:10000; Abcam), rabbit anti‐Vimentin (1:2000; Abcam), rabbit anti‐Fibronectin (1:1000; Abcam), rabbit anti‐p53 (1:1000; Abcam), rabbit anti‐MDM2 (1:1000; Abcam), rabbit anti‐Bax (1:1500; Abcam), rabbit anti‐Slug (1:1000; Abcam), rabbit anti‐Snail1 (1:1000; Abcam), rabbit anti‐Twist (0.5 μg/mL, Abcam) and rabbit anti‐GAPDH (1:500; Abcam) at 4°C overnight. Next, membranes were incubated overnight with an Horseradish Peroxidase (HRP)‐conjugated goat anti‐rabbit IgG (1:10000; Abcam) for 1 hour at 37°C. The membranes were developed with an Emitter‐Coupled Logic (ECL) chemiluminescence detection kit and quantitated using ImageJ software. GAPDH served as a loading control.