The cell layers were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) NP-40, 1 mM EDTA) supplemented with Protease inhibitor cocktail (Merck Group). Protein concentration was determined by using the Bicinchonic Protein assay kit (Euroclone S.p.A.) and 20–30 µg was resolved on SDS-polyacrylamide gels and electro-transferred to Amersham™ Hybond™ PVDF membrane (Euroclone S.P.A.). Blots were incubated o.n. at 4 °C with monoclonal anti α-smooth muscle actin (α-SMA) antibody (clone1A4) (Merck group), re-probed with anti H3 Histone antibody (Santa Cruz Biotechnology, DBA Italia s.r.l),) or p44/42 MAPK (R&D System, Bio-Techne s.r.l., Milan, Italy) and then incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology, Euroclone s.p.a., Milan, Italy). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck Group) and band intensities were determined using Alliance imaging system (Uvitec, Cambridge, UK).
Alliance imaging system
Manufactured by Uvitec
Sourced in United Kingdom, United States
The Alliance imaging system is a specialized laboratory equipment designed for capturing and analyzing images. Its core function is to provide high-quality imaging capabilities for various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Bicinchonic protein assay kit, by Euroclone (2 mentions)
Hybond, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
β tubulin, by Abcam (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Horse anti mouse, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti nox4, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Ecl chemiluminescent substrate, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Skim milk powder, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Pitpnc1, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Immunoway (1 mentions)
7 protocols using alliance imaging system
1
Western Blot Analysis of Smooth Muscle Actin
2
Western Blotting Analysis of Protein Expression
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Western blotting was carried out as described previously (13 (link)). The protein was extracted from the cells using RIPA lysis buffer with protease inhibitor cocktail (Roche), and the concentration was measured using the Poerce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). Proteins were separated by SDS-PAGE, transferred onto PVDF membranes and subsequently blocked in 5% skimmed milk. Next, the membranes were incubated with primary antibodies and secondary antibodies in order. Bands were visualized using ECL kit (EMD Millipore) and detected by the Alliance Imaging system (Uvitec, Cambridge, UK).
The following antibodies were used: C19orf10 (1:1000, PROTEINTECH, Inc), β-tubulin (1:5000, Abcam, Cambridge, UK), PTEN (1:1000, Cell Signaling Technology, Inc.), ZO-1 (1:1000, Cell Signaling Technology, Inc.), Akt (1:1000, ZENBIO.), phospho-Akt (Ser473) (1:1000, Cell Signaling Technology, Inc.), phospho-Akt (Thr308) (1:1000, Cell Signaling Technology, Inc.), goat anti-rabbit (1:1,000, cat no. sc-2004, Santa Cruz Biotechnology, Inc.), horse anti-mouse (1:1,000, cat no. 7076P2, Cell Signaling Technology, Inc.).
The following antibodies were used: C19orf10 (1:1000, PROTEINTECH, Inc), β-tubulin (1:5000, Abcam, Cambridge, UK), PTEN (1:1000, Cell Signaling Technology, Inc.), ZO-1 (1:1000, Cell Signaling Technology, Inc.), Akt (1:1000, ZENBIO.), phospho-Akt (Ser473) (1:1000, Cell Signaling Technology, Inc.), phospho-Akt (Thr308) (1:1000, Cell Signaling Technology, Inc.), goat anti-rabbit (1:1,000, cat no. sc-2004, Santa Cruz Biotechnology, Inc.), horse anti-mouse (1:1,000, cat no. 7076P2, Cell Signaling Technology, Inc.).
3
Western Blot Analysis of Apoptotic Markers
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The cell layers were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] NP-40, 1 mM EDTA, 2.5 mM DTT, 10 µg/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF, and Na3VO4). Protein concentration was determined by using the Bicinchonic Protein assay kit (Euroclone S.p.A., Milan Italy) and 20 µg were resolved on SDS-polyacrylamide gels and electro-transferred to a PVDF membrane (Millipore Billerica, MA, U.S.A.). Blots were probed using antihuman BAX (Santa Cruz Biotechnology, Santa Cruz, CA, USA), antihuman XIAP (MBL, MA, USA), anti Nox 4 (Santa Cruz Biotechnology) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology), Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Millipore Billerica, MA, U.S.A.). Band intensities were determined using Alliance imaging system (Uvitec, Cambridge, U.K.).
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted from the cells using RIPA lysis buffer (Beyotime, China). The protein concentration was quantified using a dual bicinchoninic acid protein assay kit (Beyotime, China). Protein electrophoresis was performed on 6% and 10% (w/v) sodium dodecyl sulfate–polyacrylamide gels (Vazyme, China). The separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore, USA) and blocked with 5% skim milk powder (Millipore) at room temperature for 2 h. Subsequently, the blocked membranes were incubated with specific primary antibodies overnight at 4 ℃, followed by incubation with secondary antibodies at room temperature for 1.5 h. The following antibodies were used: PITPNC1 (1:800, Sigma, USA), FASN (1:1200, Proteintech, China), CD155 (1:1000, CST, USA), Tubulin (1:5000, Proteintech, China), BCL-2 (1:1000, Abcam, USA), Cleaved Caspase-3 (1:1000, Abcam, USA), Bax (1:1000, Immunoway, USA), and goat anti-rabbit and goat anti-mouse secondary antibody (1:10000, Fude, China). After washing three times for 5 min each with TBST, the proteins were visualized using an ECL chemiluminescent substrate (Millipore, USA) on an Alliance imaging system (UVITEC, UK), and the densities were quantified using ImageJ software. Tubulin served as the internal reference protein.
5
Western Blot Analysis of DEGS1
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For total lysate preparation, washed cell pellets were resuspended in PBS containing HALT protease inhibitor cocktail (Thermo Fisher Scientific) and then brought to RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Nonidet-P40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl) containing protease inhibitor cocktail, and kept on ice with frequent vortexing for 1 h. After spinning at 12,000 g for 10 min at 4°C, the clean supernatant was removed and stored at −80°C. Aliquots of protein extracts (5–60 μg of protein) were separated by 12% SDS-PAGE, transferred to a nitrocellulose membrane using Trans-Blot SD Semi Dry Transfer Cell (Bio-Rad Laboratories) and blotted with rabbit polyclonal anti-DEGS1 (Abcam anti-MLD antibody ab167169, 1:15,000), or anti-HaloTag (Promega, 1:2000), or mouse monoclonal anti β-actin (Sigma, 1:5000), followed by secondary peroxidase labeled anti rabbit or anti mouse secondary antibody, respectively, according to our published protocol (22 (link)). Chemiluminescence was revealed using Alliance imaging system (Uvitec).
6
Western Blot Protein Analysis Protocol
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Western blot analysis was performed as described previously [44 ]. In short, cells were treated using lysing buffer (Biorad, Hercules, CA, USA) and the lysates were boiled in loading buffer for 10 min. Equal amounts of protein were separated by 8–12% sodium dodecyl sulphate–polyacrylamide gel and transferred onto nitrocellulose membranes ((Biorad, Hercules, CA, USA). The blots were blocked using 5% non-fat dry milk in TBS plus 0.05% Tween 20 and incubated with the primary antibody (Supplementary Table S1 ) overnight at 4 °C, followed by 1 h incubation at room temperature with the secondary antibody (Table S1 ) conjugated to horseradish peroxidase. Detection was carried out using the chemiluminescence (ECL) reaction (Biorad, Hercules, CA, USA) in Uvitec Alliance imaging system (Uvitec, Cambridge, UK). All immunoblots were performed in duplicates.
7
Protein Expression Analysis in HeLa Cells
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HeLa cells were incubated in the presence of the test compound and, after different times, were collected, centrifuged, and washed two times with ice cold phosphate buffered saline (PBS). The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates were centrifuged at 15000 x g at 4 °C for 10 min. The protein concentration in the supernatant was determined using the BCA protein assay reagents (Pierce, Italy). Equal amounts of protein (10 μg)
were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Criterion Precast, BioRad, Italy) and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were blocked with a bovine serum albumin solution (5% in Tween PBS 1X), the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against caspase-9 cleaved fragment, PARP, cdc25c, cyclin B, p-cdc2 Y15 (all from Cell Signaling), or vinculin (Sigma-Aldrich) for 2 h at room temperature. Membranes were next incubated with peroxidase labeled secondary antibodies for 1 h. All membranes were visualized using ECL Select (GE Healthcare), and images were acquired using a Uvitec-Alliance imaging system (Uvitec, Cambridge, UK). To ensure equal protein loading, each membrane was stripped and reprobed with an anti-vinculin antibody.
were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Criterion Precast, BioRad, Italy) and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were blocked with a bovine serum albumin solution (5% in Tween PBS 1X), the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against caspase-9 cleaved fragment, PARP, cdc25c, cyclin B, p-cdc2 Y15 (all from Cell Signaling), or vinculin (Sigma-Aldrich) for 2 h at room temperature. Membranes were next incubated with peroxidase labeled secondary antibodies for 1 h. All membranes were visualized using ECL Select (GE Healthcare), and images were acquired using a Uvitec-Alliance imaging system (Uvitec, Cambridge, UK). To ensure equal protein loading, each membrane was stripped and reprobed with an anti-vinculin antibody.
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