For whole cell lysate analysis, proteins were extracted from THP-1 and transfected HEK293T cells using lysis buffer (radioimmunoprecipitation assay, 1% protease inhibitor, 1% phosphatase inhibitor). Bolt LDS Sample Buffer (4×; Novex Life Technologies) and Bolt Sample Reducing agent (10×; Novex Life Technologies) were added to protein lysates, samples resolved on 4–12% Bis-Tris Plus gels (Invitrogen), and then transferred to nitrocellulose membrane (Invitrogen). Where protein phosphorylation status was investigated, membranes were blocked in LI-COR buffer and primary phospho-antibodies incubated for 48 h in the blocking solution. Otherwise, membranes were blocked with 5% nonfat milk in TBS and primary antibodies incubated overnight in blocking buffer supplemented with 0.1% Tween. A list of antibodies used in this study is supplied in Table S6 . Membranes were washed and incubated with appropriate anti-mouse or anti-rabbit secondary antibodies for 45 min at room temperature (LI-COR). Signal was detected using the Odyssey CLx System (LI-COR). For STING Δ1-136 revelation in coIP assays, Quick Western Blot kit (LI-COR) was used to label STING antibody and avoid anti-FLAG light chain signal, according to the manufacturer’s instructions. Comparative signal analyses were performed using Image Studio Lite (LI-COR).
Anti mouse or anti rabbit secondary antibodies
Manufactured by LI COR
Sourced in United States
Anti-mouse or anti-rabbit secondary antibodies are laboratory reagents used to detect the presence of primary antibodies that target mouse or rabbit proteins. These secondary antibodies are conjugated with reporter molecules, such as fluorescent dyes or enzymes, which allow for the visualization and quantification of the target proteins in various experimental techniques, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx system, by LI COR (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
0.45 m nitrocellulose membrane, by Cytiva (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mouse anti gapdh antibody, by Thermo Fisher Scientific (1 mentions)
Anti lc3b antibody, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
E cadherin, by Proteintech (1 mentions)
Odyssey infrared imaging system, by Gene Tech (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
4 protocols using anti mouse or anti rabbit secondary antibodies
1
Western Blot Analysis of Phosphorylated Proteins
2
Immunoblotting Protocol for Protein Detection
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Samples were loaded onto 10% or 6% SDS-PAGE gels and transferred to a 0.45-µm nitrocellulose membrane (Amersham GE Healthcare) overnight at 4°C in Tris-glycine buffer under constant voltage (30 V). Membranes were blocked with 5% nonfat dry milk and 1% BSA in PBS for 1 h at room temperature. Membranes were washed with PBS-T (PBS/0.1% Tween 20) and incubated with primary antibodies in PBS-T buffer containing 2% BSA and 0.025% sodium azide for 2 h at room temperature or overnight at 4°C. Subsequently, membranes were washed with PBS-T and incubated with anti-mouse or anti-rabbit secondary antibodies (LI-COR) diluted in antibody dilution buffer for 1 h at room temperature before scanning with an Odyssey infrared imaging system (Odyssey; LI-COR).
3
Evaluation of Autophagy Markers by Western Blotting
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Western blotting was performed to evaluate the autophagy markers. Briefly, cell lysates were prepared according to the following protocol. First, cells were incubated in RIPA buffer [Tris-HCl (pH 7.4), NaCl (150 mM), NP40 (1%), Na-deoxycholate (0.25%), EDTA (1 mM), dithiothreitol (1 mM), and phenylmethylsulfonyl fluoride (1 mM)] with a freshly added protease inhibitor cocktail (Sigma Aldrich). Thereafter, the cell lysates were centrifuged at 12.000 rpm for 10 min and the resulting supernatants were collected for western blot analysis. The protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), and 30 μg of protein per sample was loaded onto a pre-cast SDS-PAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific). The proteins were then transferred to PVDF membranes which were incubated overnight with a rabbit polyclonal anti-LC3B antibody (Abcam, Cambridge, UK) while a mouse anti-GAPDH antibody (Thermo Fisher) was used as a loading control. After washing, the membranes were incubated with fluorescently labeled anti-mouse or anti-rabbit secondary antibodies obtained from LI-COR Biosciences (Lincoln, NE, USA). The blots were scanned and imaged using the LI-COR Biosciences Odyssey® imaging system.
4
Western Blot Analysis of EMT Markers
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For western blot analysis, total protein samples were extracted from SKOV3, A2780 or OVCAR3 cells and tumor tissues collected from nude mice using RIPA lysis buffer with a protease inhibitor (Beyotime, Jiangsu, China). An equal amount of protein (60 μg/lane) was loaded on 8% SDS-polyacrylamide gels and then transferred to a Pure Nitrocellulose Blotting membrane (Pall Life Science, AZ, USA). Next, the membrane was blocked in 5% non-fat milk for 1.5 h and then probed with primary antibodies against ZEB1 (1:1000, Abcam, Cambridge, USA), E-cadherin (1:500, Proteintech, Rosemont, IL, USA), vimentin (1:1000, Cell Signaling Technology, Beverly, MA), fibronectin 1 (FN1, 1:500, Proteintech, Rosemont, IL, USA), and GAPDH (1:2000, Kangchen, Shanghai, China) overnight at 4 °C. Then, the membranes were incubated with anti-mouse or anti-rabbit secondary antibodies (1:8000; LI-COR Biosciences). Finally, immunoreactivity was detected using the Odyssey Infrared Imaging System (Gene Company Limited, Hong Kong, China).
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