Hep3b cells were washed in PBS and lysed using the protein extraction reagent RIPA (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of proteins was measured by BCA kit (cat. no. ab102536; Abcam). Equivalent amounts of proteins (30 µg) from each sample were electrophoresed on SDS-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, blocked in 4% skim milk for 2 h at room temperature and incubated with the following specific primary antibodies: E-cadherin antibody (ab219332 1:1,000; Abcam), α-catenin antibody (ab51032 1:2,000; Abcam), N-cadherin (ab76011, 1:5000 dilution, Abcam), vimentin (ab92547 1:1,000; Abcam), p-PI3K (ab182651 1:1,000; Abcam), PI3K (ab227204 1:1,000; Abcam), p-AKT (ab38449, 1:500 dilution, Abcam), AKT (ab18785 1:1,000; Abcam), VEGF (ab214424 1:1,000; Abcam), VEGFR2 (ab221679 1:1,000; Abcam), Snail (ab53519 1:1,000; Abcam), Slug (ab27568 1:1,000; Abcam) and MPP9 (ab38898 1:1,000; Abcam) overnight at 4°C. β-actin (ab8277 1:1,000; Abcam) was used as internal reference. Then, the membranes were incubated in HRP-linked goat anti-rabbit IgG secondary antibody (ab97051; 1:10,000; Abcam) for 2 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an ECL substrate buffer (EMD Millipore) and membranes were scanned with Gel Doz EZ imager (Bio-Rad Laboratories, Inc.).
Gel doz ez imager
Manufactured by Bio-Rad
The Gel Doc EZ Imager is a compact and easy-to-use gel documentation system designed for imaging and analyzing DNA, RNA, and protein gels. It provides high-quality images and allows for quick and efficient documentation of electrophoresis results.
Automatically generated - may contain errors
Lab products found in correlation
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab214424, by Abcam (1 mentions)
Ab53519, by Abcam (1 mentions)
Ab27568, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab219332, by Abcam (1 mentions)
Ab51032, by Abcam (1 mentions)
Ab8277, by Abcam (1 mentions)
Ab221679, by Abcam (1 mentions)
Ab227204, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ecl substrate buffer, by Merck Group (1 mentions)
Ab18785, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
3 protocols using gel doz ez imager
1
Western Blot Analysis of EMT and Angiogenesis Markers
2
Western Blot Analysis of Protein Expressions
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Protein expression levels were analyzed via western blot. Cells were washed twice with PBS and lysed with RIPA buffer. The protein concentration was detected using a BCA Kit (Beyotime). Equal amounts of protein (30 μg) were separated using 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C.
The specific primary antibodies were rabbit anti-Rab21 (Abcam; 1:1000 dilution), rabbit anti-Caspase7 (Abcam; 1:500 dilution), rabbit anti-Bim (Abcam; 1:500 dilution), rabbit anti-Bax (Santa Cruz Biotechnology, 1:200 dilution) and mouse anti-actin (Cell Signaling Technology; 1:1500 dilution).
The membranes were then washed with Tris-buffered and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime; 1:1000 dilution) for 1 h at room temperature. Immunoreactivity was visualized using a Millipore Enhanced Chemiluminescence system. Membranes were scanned with a Bio-rad Gel Doz EZ imager.
The specific primary antibodies were rabbit anti-Rab21 (Abcam; 1:1000 dilution), rabbit anti-Caspase7 (Abcam; 1:500 dilution), rabbit anti-Bim (Abcam; 1:500 dilution), rabbit anti-Bax (Santa Cruz Biotechnology, 1:200 dilution) and mouse anti-actin (Cell Signaling Technology; 1:1500 dilution).
The membranes were then washed with Tris-buffered and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime; 1:1000 dilution) for 1 h at room temperature. Immunoreactivity was visualized using a Millipore Enhanced Chemiluminescence system. Membranes were scanned with a Bio-rad Gel Doz EZ imager.
3
Protein Fiber Analysis by SDS-PAGE
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SDS-PAGE was performed according to the method of Laemmli, 24 with some modifications, using a 5% stacking gel (pH 6.8) and 12% separating gel (pH 8.8). The fiber samples were sheared and ground to powder. Approximately 3 mg of protein fiber powder was dissolved in 1 mL of 2X loading buffer. The mixtures were subsequently boiled for 5 min in water and centrifuged at 10,000 rpm for 5 min before loading. The electrophoresis voltage of the stacking gel and separating gel was set to 80 and 120 V, respectively. Standard protein markers with molecular weights ranging from 14.4 to 97.4 kDa were purchased from Beijing Solarbio Science & Technology Co., Ltd. After electrophoresis, the gels were rinsed with distilled water and stained with Coomassie Brilliant Blue R-250 and periodic acid-Schiff (PAS) reagent, respectively. The gels were then photographed using a Bio-rad Gel Doz EZ imager.
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