Western blotting analysis was performed as usually reported [34 (link)]. The GIST-T1 and GIST-T1/IMR cells were challenged with rabbit polyclonal anti-S1P (ab59870), anti-SPK1 (ab260073), anti-β-actin antibodies (ab8826) (Abcam company) and ABCC1 (DF7148) (Affinity company). Proteins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions (Pierce, Rockford, Illinois).
Ab8826
Manufactured by Abcam
Sourced in United Kingdom, China
Ab8826 is a primary antibody produced in rabbit. It is designed for use in immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ab260073, by Abcam (1 mentions)
Df7148, by Affinity Biosciences (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab7166, by Abcam (1 mentions)
Ab302958, by Abcam (1 mentions)
Ab278545, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab76055, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab2413, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab14734, by Abcam (1 mentions)
Ab97057, by Abcam (1 mentions)
Ab14730, by Cell Signaling Technology (1 mentions)
P0448, by Agilent Technologies (1 mentions)
Ab109865, by Abcam (1 mentions)
P0447, by Agilent Technologies (1 mentions)
6 protocols using ab8826
1
Western Blot Analysis of GIST-T1 Cells
2
Western Blot Analysis of Integrin and PI3K/AKT Signaling
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Cell pellets were lysed by adding RIPA Lysis Buffer (Beyotime, Beijing, China), and an equal amount of protein was boiled with loading buffer for 7min. Then, protein was separated by SDS–PAGE, and transferred onto polyvinylidene difluoride (PVDF). The PVDF membrane was blocked with 5% BSA and incubated overnight at 4 °C with mouse monoclonal anti-integrin αvβ3 (1:500, ab7166, Abcam, UK), rabbit monoclonal p-PI3K (1:500, ab278545, Abcam, Cambridge, UK), rabbit monoclonal total-PI3K (1:500, ab302958, Abcam, Cambridge, UK), rabbit polyclonal p-AKT (1:500, ab38449, Abcam, Cambridge, UK), rabbit polyclonal total-AKT (1:500, Abcam, ab8805, Cambridge, UK) and mouse monoclonal actin (1:500, ab8826, Abcam, Cambridge, UK). Next day, the sample was incubated with horse radish peroxidase (HRP) conjugated secondary antibody (1:1000, Abcam, Cambridge, UK) for 1 h at room temperature and then visualized by the chemiluminescence (ECL) Detection Kit (CST, Boston, MA, USA). In this study, all uncropped blotting figures were deposited in supplementary materials .
3
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA lysis buffer. The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher, USA). The supernatant was collected after centrifugation. Protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore, USA). After blocking with 5% BSA, the membrane was incubated with a primary antibody or anti-β-actin antibody as the loading control at 4 °C overnight. The following primary antibodies were used: anti-E-cadherin (1:1000, ab76055, Abcam), anti-α-smooth muscle actin (α-SMA, 1:1000, ab32575, Abcam), anti-fibronectin (1:500, ab2413, Abcam), anti-collagen IV (1:1000; Abcam), anti-plasminogen activator inhibitor-1 (PAI-1, 1:1000, ab241696, Abcam), and anti-β-actin (1:1000, ab8826, Abcam). After washing with TBST, the membrane was incubated at room temperature for 1 h with a secondary antibody. Protein bands were then visualized using hypersensitive ECL. The blots were cut before hybridization with antibodies according to different molecular weight. Band density was analyzed using ImageJ software.
4
Subcellular Fractionation and Immunoblotting
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Lysates were either made from mitochondrial fractions and whole cells. Fractions were collected in sucrose buffer (50 mM sucrose, 10 mMKCl, 20 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA [Sigma, E8145], protease inhibitor [Roche, 4693132001], pH 7.4) on ice. To break the plasma membrane, the cell suspension was passed through a 26-gauge needle (Sigma, Z192392) ~30 times. Separation of the mitochondrial and cytoplasmic fractions was performed using differential centrifugation, as shown previously [60 (link)]. Primary antibodies: ACTB (Abcam, ab8826) 1:5000, ATP5B (Abcam, ab14730) 1:5000, ERK1/2 (Cell signalling, 9102S) 1:1000, pERK1/2 (Y202/Y204) (Cell signalling, 9101) 1:500, SDHA (Abcam, ab109865), TSPO (Abcam, ab109497) 1:4000, USP30 (Enzo, MBL-PW0975) 1:1000, VDAC1 (Abcam, ab14734) 1:2000, 1:10000 Vinculin (Abcam, ab129002) and 1:1000 TFEB (Cambridge Bioscience, A303-673A). Imaging was carried using an ECL based method (GE, RPN2133) on a a Bio-Rad ChemiDoc MP Imaging System, following incubation for 1 h with either anti-rabbit conjugated HRP 1:4000 (Dako, P0447), or rabbit anti-mouse conjugated HRP (Dako, P0448), or goat anti-rat IgG conjugated HRP 1:4000 (Abcam, ab97057). Densitometric analysis was performed using the ImageJ software. Values were normalised to ACTB loading control for whole-cell lysates and ATP5B for mitochondrial fractions.
5
Western Blot Analysis of Cellular Proteins
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The total protein of TC cells was extracted with RIPA lysate (Beyotime, Shanghai, China), and the protein samples were boiled for 5 min for denaturation. The total protein was then prepared by SDS-PAGE. The protein was then transferred to PVDF membrane (Millipore, Bedford, MA, USA), which was washed with TBST, and blocked by 3% bovine serum albumin (BSA). After that, primary antibodies including anti-PPAT antibody (1: 1000, MA5-25,978, Invitrogen, Carlsbad, CA, USA), anti-PKM2 antibody (1: 1000, ab137852, Abcam, Shanghai, China), and anti-β-actin antibody (1: 2000, ab8826, Abcam, Shanghai, China) were loaded to incubate the membranes at 4°C for 8 h. After that, the membrane was washed 3 times with TBST for 10 min each time. Subsequently, HRP-labeled secondary antibody (ab205718, 1:2000, Abcam, Shanghai, China) was loaded and the PVDF membrane was incubated for 2 h. After that, ECL chemiluminescence kit (Millipore, Bedford, MA, USA) was used for showing the bands.
6
Protein Expression Analysis of Tail Tissue
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Tail tissues (3 mm) before the injury site were excised and frozen in liquid nitrogen and stored at -80 °C. Protein samples were extracted and adjusted to identical concentration. Protein mixtures were loaded onto different percentage SDS-polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Primary antibodies used were as follows: Akt (1:1000, 4685, Cell Signaling Technology (CST)), p-Akt Ser473 (1:1000, 4060, CST), p-Akt Thr308 (1:1000, 13038 S, CST), AMPKα (1:1000, 5832, CST), p-AMPKα Thr172 (1:1000, 2535, CST), mTOR (1:1000, 2983, CST), p-mTOR (1:1000, 2976, CST, 2976), p-p70 S6K Thr389 (1:1000, 9205, CST), p70 S6K (1:1000, 2708, CST), LEF1 (1:1000, ab85052, Abcam), β-catenin (1:1000, ab32572, Abcam), WE6 (1:5, WE6-s, Developmental Studies Hybridoma Bank) and β-actin (1:2000, ab8826, Abcam). β-actin was used as an internal control.
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