Las 1000 plus luminescent image analyzer

Cells were lysed with NP-40 lysis buffer, and protein concentration was determined by BCA protein assay reagent (Thermo Fisher Scientific Inc., Rockford, IL). A total of 25 μg of total protein were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerca, MA). Protein detection was conducted by the SignalBoost^TM Immunodetection Enhancer kit (Calbiochem, San Diego, CA) according to the manufacturer's recommendation. Briefly, the primary antibody was diluted with a primary antibody solution and incubated with the PVDF membrane at 4°C overnight. After washing with 0.1% Tween-20 in a Tris buffer solution, the membrane was incubated for 1 hour at room temperature with a secondary antibody that was diluted with secondary antibody buffer. The signals were developed using an ECL-plus chemiluminescence substrate (Perkin-Elmer, Waltham, MA) and captured using a LAS-1000plus Luminescent Image Analyzer (GE Healthcare Biosciences, Piscataway, NJ). The band intensity was quantified using Bio1D software (Vilber Lourmat, Marne-la-Vallée, France).

Cells were lysed in NP-40 buffer and protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples were separated by 10% SDS-PAGE and wet-transferred to a PVDF membrane (Millipore, Billerca, MA, USA) followed by incubation with primary antibodies against E-cadherin (Santa Cruz. Biotechnology Inc., Santa Cruz, CA, USA; 1:500), vimentin (Santa Cruz; 1: 500), FN-1 (Santa Cruz; 1: 500), SNAIL1 (Cell Signaling Technology Inc., Danver, MA, USA; 1: 500), TWIST (GeneTex, Irvine, CA, USA; 1: 500), ZEB1(Santa Cruz; 1: 500) or GAPDH (GeneTex, Irvine, CA, USA; 1: 5000). After incubation with corresponding secondary antibodies, the immunoreactive bands were developed using an ECL-plus chemiluminescence substrate (Perkin-Elmer, Waltham, MA, USA) and captured by LAS-1000 plus Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA) [55 (link)].

Protein samples were extracted from the BMFs or fBMFs. After denaturation, equal amounts of extracted proteins (30 μg) were separated by 10% SDS-PAGE with Tris-Glycine SDS running buffer and transferred with transfer buffer onto a nitrocellulose membrane (GE 446 Healthcare, Little Chalfont, Buckinghamshire, UK). Then, transferred membranes were incubated with BlockPRO blocking buffer (visual protein, energenesis biomedical co. ltd). After washing with TBST buffer (20 mM Tris, 150 mM NaCl and 0.1% Tween 20; pH 7.4) for 5 min 3 times, the transferred membranes were incubated with the primary anti-bodies against α-SMA (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), COL1A1(1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or GADPH (1:5000 dilution; Thermo Fisher Scientific, Waltham, MA, USA). Then, transferred membranes were washed with TBST buffer for 5 min 3 times followed by incubation with the corresponding secondary antibodies. Developed chemiluminescence signals from catalyzed ECL substrate (Pierce Biotechnology) were detected using the LAS-1000 plus Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA). After the intensity of each band was measured by densitometry, the relative intensities were calculated by normalizing to GAPDH.