Ecl plus chemiluminescence substrate

The Western blot test was carried out in accordance with the procedure previously described [62 (link)]. Antibodies to phospho-p65 markers (p-p65) were employed as primary antibodies. Primary antibodies used included antibodies to phospho-p65 markers (1:500; cat. no. sc-134306; mouse monoclonal) from Santa Cruz Biotechnology, Dallas, TX, USA, and anti-GAPDH (Millipore, Bedford, MD, USA) was used as the loading control. Following blocking, the membranes were incubated with indicated primary antibodies followed by corresponding secondary antibodies. The immunoreactive bands were generated with an ECL-plus chemiluminescence substrate (Perkin-Elmer, Waltham, MA, USA) and taken with ImageQuant LAS 4000 Mini (GE Healthcare, Piscataway, NJ, USA). Each densitometric value was expressed as the mean ± standard deviation.

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)].

Cell lysates were subjected to SDS-PAGE and transferred to the PVDF membrane (Amersham, Arlington Heights, IL, USA) by a wet-transfer. Membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies used included anti-α-SMA (Cat. #A5228), anti-COL1A1 (Cat. #C2456), anti-Smad2, and anti-phosphorylated Smad2 (Cat. #ZRB04953) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following incubation of primary antibodies, the membranes were rinsed three times and incubated with corresponding secondary antibodies. The immunoreactive bands were developed using an ECL-plus chemiluminescence substrate (Perkin-Elmer, Waltham, MA, USA) and detected by the LAS-1000 plus Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA).

Cell protein extraction and immunoblotting analysis were conducted to examine the expression of CCNG2 as previously described [51 (link)]. Th sample was boiled and separated on 10% SDS-PAGE. The proteins were transferred to PVDF membrane (Amersham, Arlington Heights, IL, USA) by wet-transfer. 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).

Mammospheres were collected by centrifugation and lysed in M-PER Mammalian Protein Extraction Reagent (Pirece Thermo Fisher Scientific Inc., Waltham, MA, USA). Then 20 μg of total protein were separated by SDS-PAGE and transferred onto PVDF membrane (Immobilon-P, Merck Millipore). The membrane was then blocked with 5% skimmed milk (Sigma-Aldrich, St. Louis, MO, USA) dissolved in Tris buffered slaine (Sigma-Aldrich) containing 0.05% Tween-20 (Sigma-Aldrich) (TBS-T) at room temperature for 1 h followed by incubation with primary antibodies at 4 °C overnight. After washing with TBS-T, the membrane was then incubated with peroxidase conjugated secondary antibodies (PerkinElmer, Waltham, MA, USA) at room temperature for one hour. The signals were developed by ECL-plus chemiluminescence substrate (PerkinElmer) and captured using a Luminescent Image Analyzer (Fusion SOLO, Vilber Lourmat, Marne-la-Vallée, France). The band intensity was quantified using ImageJ software (version 1.48a, NIH, Bethesda. MA, USA, 2013).

Cells were lysed in NP-40 buffer and protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples (25 μg of total protein) were separated by 10% SDS-PAGE and wet-transferred to a PVDF membrane (Millipore, Billerca, MA, USA). The membrane was incubated with primary antibodies recognizing Oct4 (#2750, Cell Signaling, Beverly, MA, USA), Nanog (#3850, Cell Signaling), Sox2 (#3579, Cell Signaling) or GAPDH (GTX627408, GeneTex, Irvine, CA, USA). Following primary antibodies, the membrane was incubated 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).

Cells were lysed with NP-40 lysis buffer, and protein concentration was determined by BCA protein assay reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA). A total of 25 μg of total protein were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerca, MA, USA). Protein detection was conducted by the SignalBoost™ Immunodetection Enhancer kit (Calbiochem, San Diego, CA, USA) 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 hr 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, USA) and captured using a LAS-1000plus Luminescent Image Analyzer (GE Healthcare Biosciences, Piscataway, NJ, USA). The band intensity was quantified using Bio1D software (Vilber Lourmat, Marne-la-Vallée, France).