SDS-PAGE and Western Blotting

Al-Tubuly, Abdulhamid

doi:10.1385/1-59259-076-4:391

Cited by 34 publications

(14 citation statements)

References 4 publications

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“…Proteins (20 μg/well) were separated on 4 to 12% precast gels (Invitrogen, Carlsbad, CA) using SDS-polyacrylamide gel electrophoresis under reducing conditions and then electrophoretically transferred onto polyvinylidene fluoride membrane (Invitrogen). Western blots were performed according to standard methods [19], which involved blocking in 5% non-fat milk and incubated with the primary antibody of interest, followed by incubation with a secondary antibody conjugated with the enzyme horseradish peroxidase. The detection of immunoreactive bands was performed by using the ECL Plus Western Blotting Detection System (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).…”

Section: Methodsmentioning

confidence: 99%

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Singh

Paul

Ramamoorthy

et al. 2013

Cellular Signalling

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Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cell primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular D-Ser levels and SR protein expression. Incubation with (S)-nicotine increased D-Ser levels, which was attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of D-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced D-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca2+ flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.

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Section: Methodsmentioning

confidence: 99%

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Singh

Paul

Ramamoorthy

et al. 2013

Cellular Signalling

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“…SW480 and LoVo cells were lysed in lysis buffer [0.5% sodium deoxycholate, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS)] containing 50 mM NaF, 5 mM EDTA, 1 mM DTT and 10 µg/ml aprotinin. Cell lysates were resolved on SDS-polyacrylamide gel electrophoresis, according to standard protocols (31). The samples were immunoblotted with primary antibodies agaisnt uPA, MMP-9, NF-κB, IKK, p-Thr172 5' AMP-activated protein kinase (AMPK), AMPK and β-actin, followed by incubation with secondary antibodies: Goat anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (catalog no.…”

Section: -(4 5-dimethylthiazol-2-yl)-2 5-diphenyl Tetrazolium Brommentioning

confidence: 99%

Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF–κB, uPA activator and MMP9

et al. 2016

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Abstract. Curcumin, an active nontoxic ingredient of turmeric, possesses potent anti-inflammatory, antioxidant and anti-cancer properties; however, the molecular mechanisms of curcumin are not fully understood. The transcription factor nuclear factor-κB (NF-κB) is key in cellular processes, and the expression/activation of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) are crucial for cell invasion. The present study investigated the hypothesis that curcumin inhibits colon cancer cell invasion by modulating NF-κB-mediated expression and activation of uPA and MMP9. Human colon cancer SW480 and LoVo cells were treated with various concentrations of curcumin. Curcumin was demonstrated to dose-dependently inhibit the adhesion and proliferation ability of LoVo and SW480 cells using Transwell and MTT assays, respectively. In addition, curcumin activated 5' AMP-activated protein kinase (AMPK) and suppressed p65 NF-κB phosphorylation, as shown by western blot analysis. Compound C, a potent AMPK inhibitor, abolished curcumin-induced inhibition of NF-κB, uPA and MMP9, suggesting that AMPK activation is responsible for curcumin-mediated NF-κB, uPA and MMP9 inhibition. The binding activity of NF-κB to DNA was examined and western blotting and quantitative polymerase reaction was performed to detect the effect of curcumin on the expression of uPA and MMP9. The present results revealed that curcumin significantly decreased the expression of uPA and MMP9 and NF-κB DNA binding activity. Furthermore, curcumin decreased the level of the p65 subunit of NF-κB binding to the promoter of the gene encoding uPA and MMP9, which suppressed transcriptional activation of uPA and MMP9. Overall, the present data suggest that curcumin inhibits colon cancer cell invasion via AMPK activation and subsequent inhibition of p65 NF-κB, uPA and MMP9. The therapeutic potential of curcumin for colon cancer metastasis required additional study.

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“…The trypsin inhibition activity peak was determined and the protein purity of the activity peak in each tube was detected with SDS-PAGE. The SDS-PAGE index was based on the literature (28). The gel concentration was 12% and Coomassie Brilliant Blue R-250 was used for staining.…”

Section: Methodsmentioning

confidence: 99%

Rhizoma Pinelliae trypsin inhibitor separation, purification and inhibitory activity on the proliferation of BGC-823 gastric adenocarcinoma cells

Hou-wei

Wang

et al. 2014

Experimental and Therapeutic Medicine

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The aim of this study was to isolate and purify Rhizoma Pinelliae trypsin inhibitor (RPTI), determine its N-terminal amino acid sequence and evaluate its inhibitory effect on the proliferation of poorly differentiated BGC-823 human gastric adenocarcinoma cells. RPTI was separated and purified from a 40% (NH4)2SO4 precipitate of crude protein extract of Pinellia ternata tuber using affinity chromatography with trypsin as the ligand. The N-terminal amino acid sequence of RPTI was determined using the Edman degradation method. The inhibitory effect of RPTI on BGC-823 cell proliferation was detected in vitro using the MTT method and in vivo in tumour-bearing mice. The purified RPTI showed a single band under SDS-PAGE, its molecular weight was 14 kDa and its N-terminal amino acid sequence was DPVVDG. RPTI inhibited trypsin activity, with an inhibition ratio of 1:6.78 (mass). RPTI significantly inhibited the proliferation of BGC-823 cells in vitro. The IC50 of RPTI was 16.96 μg/ml within 48 h after treatment and 9.61 μg/ml within 72 h after treatment. Subcutaneous injection of RPTI around the tumour significantly inhibited BGC-823 tumour growth in mice. The tumour inhibitory effect was concentration- and dose-dependent. RPTI did not significantly influence the spleen coefficient of the mice. In conclusion, RPTI is a serine proteinase inhibitor with antitumour activity.

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SDS-PAGE and Western Blotting

Cited by 34 publications

References 4 publications

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF–κB, uPA activator and MMP9

Rhizoma Pinelliae trypsin inhibitor separation, purification and inhibitory activity on the proliferation of BGC-823 gastric adenocarcinoma cells

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