ผลต่างระหว่างรุ่นของ "หน้าหลัก"
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− | + | Emented with 10 fetal bovine serum, penicillin and streptomycin. 786-0 (WT) and | |
+ | Emented with ten fetal bovine serum, penicillin and streptomycin. 786-0 (WT) and 786-0 (pRC), developed by steady transfection of with wild-type VHL or empty pRC vector, respectively, had been a present from W. Kaelin (16). They have been chosen with G418 (500ug/mL) just about every sixth passage. RCC4 was generouslyCancer Res. Author manuscript; accessible in PMC 2015 July 01.Gregg et al.Pageprovided by M. C. Simon and Caki1 cells had been obtained from ATCC. Cell lines have been routinely authenticated by DNA fingerprinting in the get started and twice annually for the duration of these studies by the core University of Pittsburgh Cancer Institute Cell Culture and Cytogenetics Facility. Steady Nox4 knockdown was accomplished for every cell line by expressing two Nox4 shRNAs or maybe a non-targeting shRNA in pSilencerTM four.1-CMV puro (Ambion, Austin, TX) as previously described.(10) Steady transfectants had been maintained in puromycin (1g/mL). RT-PCR for Nox1-5, p22phox, p47phox and p67phox was performed as described (17). Adenoviral vectors Ad-EGFP, Ad-MnSOD and Ad-catalase had been a generous present of Dr. Yong Lee (18). Adenoviral transduction was performed as previously described (19). Briefly, cells had been infected at one hundred or 200 MOI for 1.5 hours in DMEM. Assays were performed 48 hours post transduction. To overexpress Nox4, parental 786-0 cells had been transfected with a pcDNA vector expressing the total human Nox4 cDNA and antibiotic selection of steady clones was performed. Cells had been pre-treated for four hours with indicated concentrations of DLDithiothreitol (DTT, Promega, Madison, WI) or 4-hydroxy-TEMPOL (Sigma-Aldrich, St. Louis, MO) before fixation or live cell assay. Drug was maintained in the media throughout reside cell assays. Quantitative RT-PCR Total RNA was extracted from 786-O, RCC4, and LNCap cells with TRIzol reagent and RNeasy Mini Kit (Qiagen, Valencia,CA). Very first strand cDNA was synthesized employing iScript cDNA synthesis kit (BIO-RAD, Hercules,CA ). Gene-specific TaqMan Gene Expression Assays primer sets and Master Mix have been utilised for quantitative PCR of NOX4 (Hs00418356), NOX1 (Hs00246589), and GAPDH (Hs99999905). Samples had been then subjected to real-time PCR evaluation working with the ABI StepOnePlus real-Time PCR Method (Applied Biosystems, Carlsbad, CA). Relative mRNA expression of each and every transcript was normalized against GAPDH. Western blot Protein was extracted as previously described (4). Equal amounts of protein were subjected to separation within a four.5?five Tris-HCl gel, and the resolved proteins had been transferred to polyvinylidene difluoride membrane. The blots were probed with anti-Nox4 rabbit monoclonal Ab (1:2,000, Abcam, Cambridge, MA) or -Actin Ab (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated secondary Ab. Bands have been visualized making use of SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Measurement of NAD(P)H oxidase activity Superoxide production from membrane fraction was determined by lucigenin chemiluminescence as described (20). Briefly, 20 g protein was added to 200 l of 50 mM phosphate buffer with 1mM EGTA, 150mM sucrose, and one hundred M NADPH. Lucigenin was added, and chemiluminescence study just about every 30 seconds for 20 min (SpectraMax Plus 384, Sunnyvale, CA) and expressed as relative light units (RLU)/mg protein. |
รุ่นแก้ไขเมื่อ 01:08, 21 กรกฎาคม 2564
Emented with 10 fetal bovine serum, penicillin and streptomycin. 786-0 (WT) and Emented with ten fetal bovine serum, penicillin and streptomycin. 786-0 (WT) and 786-0 (pRC), developed by steady transfection of with wild-type VHL or empty pRC vector, respectively, had been a present from W. Kaelin (16). They have been chosen with G418 (500ug/mL) just about every sixth passage. RCC4 was generouslyCancer Res. Author manuscript; accessible in PMC 2015 July 01.Gregg et al.Pageprovided by M. C. Simon and Caki1 cells had been obtained from ATCC. Cell lines have been routinely authenticated by DNA fingerprinting in the get started and twice annually for the duration of these studies by the core University of Pittsburgh Cancer Institute Cell Culture and Cytogenetics Facility. Steady Nox4 knockdown was accomplished for every cell line by expressing two Nox4 shRNAs or maybe a non-targeting shRNA in pSilencerTM four.1-CMV puro (Ambion, Austin, TX) as previously described.(10) Steady transfectants had been maintained in puromycin (1g/mL). RT-PCR for Nox1-5, p22phox, p47phox and p67phox was performed as described (17). Adenoviral vectors Ad-EGFP, Ad-MnSOD and Ad-catalase had been a generous present of Dr. Yong Lee (18). Adenoviral transduction was performed as previously described (19). Briefly, cells had been infected at one hundred or 200 MOI for 1.5 hours in DMEM. Assays were performed 48 hours post transduction. To overexpress Nox4, parental 786-0 cells had been transfected with a pcDNA vector expressing the total human Nox4 cDNA and antibiotic selection of steady clones was performed. Cells had been pre-treated for four hours with indicated concentrations of DLDithiothreitol (DTT, Promega, Madison, WI) or 4-hydroxy-TEMPOL (Sigma-Aldrich, St. Louis, MO) before fixation or live cell assay. Drug was maintained in the media throughout reside cell assays. Quantitative RT-PCR Total RNA was extracted from 786-O, RCC4, and LNCap cells with TRIzol reagent and RNeasy Mini Kit (Qiagen, Valencia,CA). Very first strand cDNA was synthesized employing iScript cDNA synthesis kit (BIO-RAD, Hercules,CA ). Gene-specific TaqMan Gene Expression Assays primer sets and Master Mix have been utilised for quantitative PCR of NOX4 (Hs00418356), NOX1 (Hs00246589), and GAPDH (Hs99999905). Samples had been then subjected to real-time PCR evaluation working with the ABI StepOnePlus real-Time PCR Method (Applied Biosystems, Carlsbad, CA). Relative mRNA expression of each and every transcript was normalized against GAPDH. Western blot Protein was extracted as previously described (4). Equal amounts of protein were subjected to separation within a four.5?five Tris-HCl gel, and the resolved proteins had been transferred to polyvinylidene difluoride membrane. The blots were probed with anti-Nox4 rabbit monoclonal Ab (1:2,000, Abcam, Cambridge, MA) or -Actin Ab (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated secondary Ab. Bands have been visualized making use of SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Measurement of NAD(P)H oxidase activity Superoxide production from membrane fraction was determined by lucigenin chemiluminescence as described (20). Briefly, 20 g protein was added to 200 l of 50 mM phosphate buffer with 1mM EGTA, 150mM sucrose, and one hundred M NADPH. Lucigenin was added, and chemiluminescence study just about every 30 seconds for 20 min (SpectraMax Plus 384, Sunnyvale, CA) and expressed as relative light units (RLU)/mg protein.