Total cellular protein was extracted from FTC-133 or 8505C cells treated with increasing concentrations of GB1107 or TD139 (0, 10, and 100 μM) for 24 h. Equal amounts of protein were electrophoresed in 10% polyacrylamide gels and transferred to the nitrocellulose membrane. The membrane was blocked in 5% skim milk, incubated with the primary antibody at 4°C overnight, and then treated with secondary antibody at 37°C for 1 h [14 (link)]. The primary antibodies used for western blotting were purchased from Cell Signaling Technology, Danvers, MA, USA, unless otherwise specified: galectin-3 (1 : 1000 dilution; MABT51; Sigma-Aldrich), cyclin D1 (1 : 700; #2978), cleaved caspase-3 (1 : 700; #9661), PARP1 (1 : 700; ab32138; Abcam), cleaved PARP1 (1 : 700; ab32064; Abcam), phospho-p44/42 MAPK (ERK1/2) Thr202/Tyr204 (1 : 700; #9101), ERK1/2 (1 : 700; #9102), phospho-p38 MAPK Thr180/Tyr182 (1 : 700; #9216), p38 (1 : 700; #9212), phospho-JNK1 Thr183/Tyr185 (1 : 700; ab215208; Abcam), JNK1 (1 : 700; ab110724; Abcam), phospho-AKT Ser473 (1 : 700; #9271), AKT (1 : 700; #4691), β-catenin (1 : 700; #9562), MMP2 (1 : 500; MAB3308; Sigma-Aldrich), and β-actin (1 : 5000; A5441; Sigma-Aldrich).
Mab3308
Manufactured by Merck Group
Sourced in United States
MAB3308 is a laboratory equipment product from Merck Group. It is a monoclonal antibody designed for use in various research applications. The core function of MAB3308 is to provide a specific and reliable detection tool for researchers.
Automatically generated - may contain errors
Lab products found in correlation
Ab32064, by Abcam (1 mentions)
Ab215208, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
Ab32138, by Abcam (1 mentions)
Ab110724, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ab61224, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ecl chemiluminescence kit, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Mab1501, by Merck Group (1 mentions)
4 protocols using mab3308
1
Protein Expression and Signaling Analysis
2
Gelatinases and Inhibitors Expression
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The protein expression of gelatinases i.e., MMP-2 & MMP-9 (Sigma-Aldrich, United States; MAB3308, MAB3309) and their respective inhibitors-TIMP-1 & TIMP-2 (Sigma-Aldrich, United States; MAB3310; Abcam, United States; ab61224) in response to cadmium treatment were analyzed by Western blotting. Equal amounts of crude protein was loaded and resolved by reducing SDS-PAGE. Separated proteins were electroblotted to polyvinylidene difluoride (PVDF) membrane (GE Healthcare Limited, Buckinghamshire). Electroblots were washed with TBST (20 mM Tris-buffered saline and 0.1% Tween-20), blocked and probed with primary antibodies for the targets overnight at 4°C. Detection was done using compatible secondary HRP-conjugated antibodies (Abcam, United States; ab205718, ab205719) and incubated in ECL solution for a minute. Immunoreactive bands were visualized using ECL (ThermoFisher-Scientific, United States; 32209) under SynGene Gel documentation system. Protein expressions was evaluated by densitometric analysis using Image studio Lite (Ver 5.2) and normalized against the corresponding expression of GAPDH. Analysis was presented as a fold change compared to the respective controls.
3
Aortic Gelatinolytic Activity and MMP-2 Co-localization
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In situ gelatinolytic activity in the media of frozen thoracic aorta was performed as previously described [28] (link). Frozen 4 μm sections were incubated with dye-quenched (DQ) Gelatin (E12055, Molecular Probes, Oregon 411, USA) diluted 1:20 for 30 min in dark humidified chambers. The images were examined with fluorescent microscopy (Leica Imaging Systems Ltd., Cambridge, England) and captured at 400×. The intensity of the green fluorescent signal was evaluated by using ImageJ Program (NIH – National Institute of Health).
To co-localized aortic gelatinolytic activity with MMP-2 expression immunofluorescence for MMP-2 was performed. After DQ gelatin, the sections were rinsed 3× with cold PBS and incubated with mouse monoclonal MMP-2 antibody (1:500; MAB3308, Millipore, USA) for 1 h. Slices were then incubated with anti-mouse rhodamine conjugated secondary antibody (1:200, AP181R, Millipore, USA) Sections were examined with fluorescent microscopy (Leica Imaging Systems Ltd., Cambridge, England) and the image was captured at 400×. The intensity of the red fluorescent signal was evaluated by using ImageJ Program [28] (link).
To co-localized aortic gelatinolytic activity with MMP-2 expression immunofluorescence for MMP-2 was performed. After DQ gelatin, the sections were rinsed 3× with cold PBS and incubated with mouse monoclonal MMP-2 antibody (1:500; MAB3308, Millipore, USA) for 1 h. Slices were then incubated with anti-mouse rhodamine conjugated secondary antibody (1:200, AP181R, Millipore, USA) Sections were examined with fluorescent microscopy (Leica Imaging Systems Ltd., Cambridge, England) and the image was captured at 400×. The intensity of the red fluorescent signal was evaluated by using ImageJ Program [28] (link).
4
Quantification of MMP-2 Expression in Aortic Tissue
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Aortic samples of about 30 mg were lysed with cold RIPA-buffer. The protein concentration of tissue homogenate was determined with Bradford protein assay. 40 μg of protein extracts were separated by 12% polyacrylamide gels. Then, the proteins were transferred to nitrocellulose membranes (GE Healthcare, Madison, WI, USA). The membranes were blocked for 1 h at room temperature with TBS-T (NaCl 100 mM; Tris–Cl 100 mM; Tween 0.1%) containing 5% BSA (bovine serum albumin) and incubated overnight at 4 °C with primary antibody against MMP-2 (1:1000; MAB3308, Millipore, USA). The membranes were then incubated with 1:1000 horseradish peroxidase (HRP)-secondary goat anti-rabbit antibody (AP132P, Millipore, USA) for 1 h. The protein bands were revealed with ECL chemiluminescence kit (GE Healthcare). MMP-2 expression was normalized with respect to β-actin expression (1:1000; MAB1501, Millipore, USA).
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