(c) Cells were cultivated for 24 h in the absence (ctrl) or presence of 75 nM Tariquidar (Tar) or 75 nM = 3). micrograms of proteins was subjected to immunoblotting and probed Ethisterone with the following antibodies: anti-ABCB1/Pgp (C219, Novus Biologicals, Littleton, CO, USA; dilution 1/250), anti-ABCC1/MRP1 (IU2H10, Abcam, Cambridge, UK; dilution 1/100), anti-ABCG2/BCRP (sc-25882, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/500), followed by a peroxidase-conjugated secondary antibody. Proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). Plasma membrane-associated proteins were evaluated in biotinylation assays, using the Cell Surface Protein Isolation kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) [15], and probed with anti-Pgp and anti-CRT (PA3-900, ABR-Affinity BioReagents Inc., Golden, CO, USA; dilution 1/500) antibodies. Non-biotinylated proteins, i.e., cytosolic proteins, were blotted with the anti-Pgp antibody. Anti–tubulin antibody (sc-5274, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/1000) was used as control of equivalent protein loading in cytosolic components; anti-pancadherin antibody (CH-19; Santa Cruz Biotechnology Inc., dilution 1/500) was used mainly because control in plasma membrane components. In co-immunoprecipitation experiments, 100 g of plasma membrane-associated proteins were immunoprecipitated with the anti-CRT antibody, using PureProteome protein A and protein G Magnetic Beads (Millipore, Bedford, MA, USA), and then blotted for Pgp. To assess Pgp ubiquitination, 50 g whole cell lysate was immunoprecipitated with the anti-Pgp antibody, and then probed with an anti-mono/polyubiquitin antibody (FK2, Axxora, Lausanne, Switzerland; dilution 1/1000). 2.4. Intracellular Doxorubicin Build up and Doxorubicin Kinetic Efflux The intracellular doxorubicin content material and the drug efflux were measured as detailed in [26]. The intracellular doxorubicin concentration was indicated as nanomoles doxorubicin/mg cellular proteins. The efflux of doxorubicin was indicated as the switch in the intracellular concentration of the drug/minute (dc/dt) [26]. Km and Vmax guidelines were estimated using the Enzfitter software (Biosoft Corporation, Cambridge, UK). 2.5. ATPases Activity Pgp, MRP1, and BCRP were immunoprecipitated from 100 g of membrane-associated proteins, then the rate of ATP hydrolysis, an index of the catalytic cycle and a necessary step for substrate efflux, was measured spectrophotometrically [27]. In each set of experiments, 0.5 mM Na3VO4 was included in the reaction mix to measure the Na3VO4-sensitive rate of ATP hydrolysis. Results were indicated as nmoles hydrolyzed phosphate/mg protein. 2.6. Caspase 3 Activity Cells were lysed in 0.5 mL of lysis buffer (20 mM Hepes/KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiotreitol DTT, 1 mM PMSF, 10 g/mL leupeptin, pH 7.5). Twenty micrograms of cell lysates was incubated for 1 h at 37 C with 20 M of the fluorogenic substrate of caspase-3 Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), in 0.25 mL of assay buffer (25 mM Hepes, 0.1% 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate CHAPS, 10% sucrose, and 10 mM DTT, 0.01% egg albumin, pH 7.5). The reaction was stopped by adding 0.75 mL of Ethisterone ice-cold 0.1% trichloroacetic acid, and the fluorescence of AMC fragment released by active caspases was go through using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Devices, Winooski, VT, USA). Excitation and emission wavelengths were 380 and 460 nm, respectively. Fluorescence was converted in nmoles AMC/mg cellular proteins, using a calibration curve prepared previously with standard solutions of AMC. 2.7. Cell Viability Cell viability was evaluated using the ATPLite kit (PerkinElmer, Waltham, MA, USA). The results were indicated as percentage of viable cells in each experimental condition versus untreated cells (regarded as 100% viable). 2.8. Proximity Ligation Assay The CRTCPgp connection was measured with the DuoLink In Situ Kit (Sigma-Merck), as per manufacturers instructions, using the mouse anti-Pgp (UIC-2, Millipore; dilution 1/50) and the rabbit anti-CRT (PA3-900, ABR-Affinity BioReagents Inc.; dilution 1/50) antibodies. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Cells were examined using a Leica DC100 fluorescence microscope (Leica Microsystem, Wetzlar, Germany). 2.9. Confocal Microscope Analysis Cells were seeded onto glass coverslips, and transduced with the CellLight Early Endosomes-GFP Reagent BacMam 2.0 (Invitrogen, Milan, Italy), containing an expression vector for green fluorescent protein (GFP)-Rab5a, according to the manufacturers instructions. Cells were then fixed using 4% paraformaldehyde.= 3). by enhanced chemiluminescence (Bio-Rad Laboratories). Plasma membrane-associated proteins were evaluated in biotinylation assays, using the Cell Surface Protein Isolation kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) [15], and probed with anti-Pgp and anti-CRT (PA3-900, ABR-Affinity BioReagents Inc., Golden, CO, USA; dilution 1/500) antibodies. Non-biotinylated proteins, i.e., cytosolic proteins, were blotted with the anti-Pgp antibody. Anti–tubulin antibody (sc-5274, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/1000) was used as control of equivalent protein loading in cytosolic components; anti-pancadherin antibody (CH-19; Santa Cruz Biotechnology Inc., dilution 1/500) was used mainly because control in plasma membrane components. In co-immunoprecipitation experiments, 100 g of plasma membrane-associated proteins were immunoprecipitated with the anti-CRT antibody, using PureProteome protein A and protein G Magnetic Beads (Millipore, Bedford, MA, USA), and then blotted for Pgp. To assess Pgp ubiquitination, 50 g whole cell lysate was immunoprecipitated with the anti-Pgp antibody, and then probed with an anti-mono/polyubiquitin antibody (FK2, Axxora, Lausanne, Switzerland; dilution 1/1000). 2.4. Intracellular Doxorubicin Build up and Doxorubicin Kinetic Efflux The intracellular doxorubicin content material and the drug efflux were measured as detailed in [26]. The intracellular doxorubicin concentration was indicated as nanomoles doxorubicin/mg cellular proteins. The efflux of doxorubicin was indicated as the switch in the intracellular concentration of the drug/minute (dc/dt) [26]. Km and Vmax parameters were estimated using the Enzfitter software (Biosoft Corporation, Cambridge, UK). 2.5. ATPases Activity Pgp, MRP1, and BCRP were immunoprecipitated from 100 g of membrane-associated proteins, then the rate of ATP hydrolysis, an index of the catalytic cycle and a necessary step for substrate efflux, was measured spectrophotometrically [27]. In each set of experiments, 0.5 mM Na3VO4 was included in the reaction mix to measure the Na3VO4-sensitive rate of ATP hydrolysis. Results were expressed as nmoles hydrolyzed phosphate/mg protein. 2.6. Caspase 3 Activity Cells were lysed in 0.5 mL of lysis buffer (20 mM Hepes/KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiotreitol DTT, 1 mM PMSF, 10 g/mL leupeptin, pH 7.5). Twenty micrograms of cell lysates was incubated for 1 h at 37 C with 20 M of the fluorogenic substrate of caspase-3 Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), in 0.25 mL of assay buffer (25 mM Hepes, 0.1% 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate CHAPS, 10% sucrose, and 10 mM DTT, 0.01% egg albumin, pH 7.5). The reaction was stopped by adding 0.75 mL of ice-cold 0.1% trichloroacetic acid, and the fluorescence of AMC fragment released by active caspases was read using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). Excitation and emission wavelengths were 380 and 460 nm, respectively. Fluorescence was converted in nmoles AMC/mg cellular proteins, using a calibration curve prepared previously with standard solutions of AMC. 2.7. Cell Viability Cell viability was evaluated using the ATPLite kit (PerkinElmer, Waltham, MA, USA). The results were expressed as percentage of viable cells in each experimental condition versus untreated cells (considered 100% viable). 2.8. Proximity Ligation Assay The CRTCPgp conversation was measured with the DuoLink In Situ Kit (Sigma-Merck), as per manufacturers instructions, using the mouse anti-Pgp (UIC-2, Millipore; dilution 1/50) and the rabbit anti-CRT (PA3-900, ABR-Affinity BioReagents Inc.; dilution 1/50) antibodies. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Cells were examined using a Leica DC100 fluorescence microscope (Leica Microsystem, Wetzlar, Germany). 2.9. Confocal Microscope Analysis Cells were seeded onto glass coverslips, and transduced with the CellLight Early Endosomes-GFP Reagent BacMam 2.0 (Invitrogen, Milan, Italy), containing an expression vector for green fluorescent protein (GFP)-Rab5a, according.Physique S4: Amount of Pgp in whole cell extracts Click here for additional data file.(294K, pdf) Author Contributions Conceptualization, E.T., S.D., M.C., and C.R.; methodology, J.K., M.G., S.D., and M.C.; validation, M.C.; investigation, J.K., M.G., R.G., D.C.B., and M.A.; data curation, J.K.; writingoriginal draft preparation, J.K. with the following antibodies: anti-ABCB1/Pgp (C219, Novus Biologicals, Littleton, CO, USA; dilution 1/250), anti-ABCC1/MRP1 (IU2H10, Abcam, Cambridge, UK; dilution 1/100), anti-ABCG2/BCRP (sc-25882, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/500), followed by a peroxidase-conjugated secondary antibody. Proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). Plasma membrane-associated proteins were evaluated in biotinylation assays, using the Cell Surface Protein Isolation kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) [15], and probed with anti-Pgp and anti-CRT (PA3-900, ABR-Affinity BioReagents Inc., Golden, CO, USA; dilution 1/500) antibodies. Non-biotinylated proteins, i.e., cytosolic proteins, were blotted with the anti-Pgp antibody. Anti–tubulin antibody (sc-5274, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/1000) was used as control of equal protein loading in cytosolic extracts; anti-pancadherin antibody (CH-19; Santa Cruz Biotechnology Inc., dilution 1/500) was used as control in plasma membrane extracts. In co-immunoprecipitation experiments, 100 g of plasma membrane-associated proteins were immunoprecipitated with the anti-CRT antibody, using PureProteome protein A and protein G Magnetic Beads (Millipore, Bedford, MA, USA), and then blotted for Pgp. To assess Pgp ubiquitination, 50 g whole cell lysate was immunoprecipitated with the anti-Pgp antibody, and Ethisterone then probed with an anti-mono/polyubiquitin antibody (FK2, Axxora, Lausanne, Switzerland; dilution 1/1000). 2.4. Intracellular Doxorubicin Accumulation and Doxorubicin Kinetic Efflux The intracellular doxorubicin content and the drug efflux were measured as detailed in [26]. The intracellular doxorubicin concentration was expressed as nanomoles doxorubicin/mg cellular proteins. The efflux of doxorubicin was expressed as the change in the intracellular concentration Ethisterone of the drug/minute (dc/dt) [26]. Km and Vmax parameters were estimated using the Enzfitter software (Biosoft Corporation, Cambridge, UK). 2.5. ATPases Activity Pgp, MRP1, and BCRP were immunoprecipitated from 100 g of membrane-associated proteins, then the rate of ATP hydrolysis, an index of the catalytic cycle and a necessary step for substrate efflux, was measured spectrophotometrically [27]. In each set of experiments, 0.5 mM Na3VO4 was included in the reaction mix to measure the Na3VO4-sensitive rate of ATP hydrolysis. Results were expressed as nmoles hydrolyzed phosphate/mg protein. 2.6. Caspase 3 Activity Cells were lysed in 0.5 mL of lysis buffer (20 mM Hepes/KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiotreitol DTT, 1 mM PMSF, 10 g/mL leupeptin, pH 7.5). Twenty micrograms of cell lysates was incubated for 1 h at 37 C with 20 M of the fluorogenic substrate of caspase-3 Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), in 0.25 mL of assay buffer (25 mM Hepes, 0.1% 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate CHAPS, 10% sucrose, and 10 mM DTT, 0.01% egg albumin, pH 7.5). The reaction was stopped by adding 0.75 mL of ice-cold 0.1% trichloroacetic acid, and the fluorescence of AMC fragment released by active caspases was read using a Synergy Rabbit Polyclonal to GLU2B HT Multi-Detection Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). Excitation and emission wavelengths were 380 and 460 nm, respectively. Fluorescence was converted in nmoles AMC/mg cellular proteins, using a calibration curve prepared previously with standard solutions of AMC. 2.7. Cell Viability Cell viability was evaluated using the ATPLite kit (PerkinElmer, Waltham, MA, USA). The results were expressed as percentage of viable cells in each experimental condition versus untreated cells (considered 100% viable). 2.8. Proximity Ligation Assay The CRTCPgp conversation was measured with the DuoLink In Situ Kit (Sigma-Merck), as per manufacturers instructions, using the mouse anti-Pgp (UIC-2, Millipore; dilution 1/50) and the rabbit anti-CRT (PA3-900, ABR-Affinity BioReagents Inc.; dilution 1/50) antibodies. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Cells were examined using a Leica DC100 fluorescence microscope (Leica Microsystem, Wetzlar, Germany). 2.9. Confocal Microscope Analysis Cells were seeded onto glass coverslips, and transduced.This resistance to doxorubicin-induced ICD was observed also in MDA-MB-231/DX cells, i.e., the doxorubicin-resistant version with the best degrees of Pgp and the cheapest retention of doxorubicin, among the cell lines examined. Surface Proteins Isolation package (Thermo Fisher Scientific Inc., Rockford, IL, USA) [15], and probed with anti-Pgp and anti-CRT (PA3-900, ABR-Affinity BioReagents Inc., Golden, CO, USA; dilution 1/500) antibodies. Non-biotinylated protein, i.e., cytosolic protein, had been blotted using the anti-Pgp antibody. Anti–tubulin antibody (sc-5274, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/1000) was utilized as control of similar proteins launching in cytosolic components; anti-pancadherin antibody (CH-19; Santa Cruz Biotechnology Inc., dilution 1/500) was utilized mainly because control in plasma membrane components. In co-immunoprecipitation tests, 100 g of plasma membrane-associated proteins had been immunoprecipitated using the anti-CRT antibody, using PureProteome proteins A and proteins G Magnetic Beads (Millipore, Bedford, MA, USA), and blotted for Pgp. To assess Pgp ubiquitination, 50 g entire cell lysate was immunoprecipitated using the anti-Pgp antibody, and probed with an anti-mono/polyubiquitin antibody (FK2, Axxora, Lausanne, Switzerland; dilution 1/1000). 2.4. Intracellular Doxorubicin Build up and Doxorubicin Kinetic Efflux The intracellular doxorubicin content material and the medication efflux had been measured as complete in [26]. The intracellular doxorubicin focus was indicated as nanomoles doxorubicin/mg mobile proteins. The efflux of doxorubicin was indicated as the modification in the intracellular focus of the medication/minute (dc/dt) [26]. Kilometres and Vmax guidelines had been approximated using the Enzfitter software program (Biosoft Company, Cambridge, UK). 2.5. ATPases Activity Pgp, MRP1, and BCRP had been immunoprecipitated from 100 g of membrane-associated protein, then the price of ATP hydrolysis, an index from the catalytic routine and a required stage for substrate efflux, was assessed spectrophotometrically [27]. In each group of tests, 0.5 mM Na3VO4 was contained in the reaction mix to gauge the Na3VO4-sensitive rate of ATP hydrolysis. Outcomes had been indicated as nmoles hydrolyzed phosphate/mg proteins. 2.6. Caspase 3 Activity Cells had been lysed in 0.5 mL of lysis buffer (20 mM Hepes/KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiotreitol DTT, 1 mM PMSF, 10 g/mL leupeptin, pH 7.5). Twenty micrograms of cell lysates was incubated for 1 h at 37 C with 20 M from the fluorogenic substrate of caspase-3 Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), in 0.25 mL of assay buffer (25 mM Hepes, 0.1% 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate CHAPS, 10% sucrose, and 10 mM DTT, 0.01% egg albumin, pH 7.5). The response was stopped with the addition of 0.75 mL of ice-cold 0.1% trichloroacetic acidity, as well as the fluorescence of AMC fragment released by dynamic caspases was examine utilizing a Synergy HT Multi-Detection Microplate Audience (Bio-Tek Tools, Winooski, VT, USA). Excitation and emission wavelengths had been 380 and 460 nm, respectively. Fluorescence was transformed in nmoles AMC/mg mobile protein, utilizing a calibration curve ready previously with regular solutions of AMC. 2.7. Cell Viability Cell viability was examined using the ATPLite package (PerkinElmer, Waltham, MA, USA). The outcomes had been indicated as percentage of practical cells in each experimental condition versus neglected cells (regarded as 100% practical). 2.8. Closeness Ligation Assay The CRTCPgp discussion was measured using the DuoLink In Situ Package (Sigma-Merck), according to manufacturers guidelines, using the mouse anti-Pgp (UIC-2, Millipore; dilution 1/50) as well as the rabbit anti-CRT (PA3-900, ABR-Affinity BioReagents Inc.; dilution 1/50) antibodies. Cell nuclei had been counterstained with 4,6-diamidino-2-phenylindole (DAPI). Cells had been examined utilizing a Leica DC100 fluorescence microscope (Leica Microsystem, Wetzlar, Germany). 2.9. Confocal Microscope Evaluation Cells had been seeded onto cup coverslips, and transduced using the CellLight Early Endosomes-GFP Reagent BacMam 2.0 (Invitrogen, Milan, Italy), containing a manifestation vector for green fluorescent protein (GFP)-Rab5a, based on the manufacturers guidelines. Cells had been then set using 4% paraformaldehyde for 15 min, cleaned with PBS, and incubated for 1.Alternatively, breast cancer individuals with high CD8+CTL infiltration, which correlated with an increased intratumor expression of CRT, have an improved success [46]. mM phenylmethylsulphonyl fluoride PMSF, pH 7.5), centrifuged and sonicated at 13,000 for 10 min at 4 C. Fifty micrograms of protein was put through immunoblotting and probed with the next antibodies: anti-ABCB1/Pgp (C219, Novus Biologicals, Littleton, CO, USA; dilution 1/250), anti-ABCC1/MRP1 (IU2H10, Abcam, Cambridge, UK; dilution 1/100), anti-ABCG2/BCRP (sc-25882, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/500), accompanied by a peroxidase-conjugated supplementary antibody. Proteins had been detected by improved chemiluminescence (Bio-Rad Laboratories). Plasma membrane-associated protein had been examined in biotinylation assays, using the Cell Surface area Protein Isolation package (Thermo Fisher Scientific Inc., Rockford, IL, USA) [15], and probed with anti-Pgp and anti-CRT (PA3-900, ABR-Affinity BioReagents Inc., Golden, CO, USA; dilution 1/500) antibodies. Non-biotinylated protein, i.e., cytosolic protein, had been blotted using the anti-Pgp antibody. Anti–tubulin antibody (sc-5274, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution 1/1000) was utilized as control of similar proteins launching in cytosolic components; anti-pancadherin antibody (CH-19; Santa Cruz Biotechnology Inc., dilution 1/500) was utilized mainly because control in plasma membrane components. In co-immunoprecipitation tests, 100 g of plasma membrane-associated proteins had been immunoprecipitated using the anti-CRT antibody, using PureProteome proteins A and proteins G Magnetic Beads (Millipore, Bedford, MA, USA), and blotted for Pgp. To assess Pgp ubiquitination, 50 g entire cell lysate was immunoprecipitated using the anti-Pgp antibody, and probed with an anti-mono/polyubiquitin antibody (FK2, Axxora, Lausanne, Switzerland; dilution 1/1000). 2.4. Intracellular Doxorubicin Build up and Doxorubicin Kinetic Efflux The intracellular doxorubicin content material and the medication efflux had been measured as complete in [26]. The intracellular doxorubicin focus was indicated as nanomoles doxorubicin/mg mobile proteins. The efflux of doxorubicin was indicated as the modification in the intracellular focus of the medication/minute (dc/dt) [26]. Kilometres and Vmax guidelines had been approximated using the Enzfitter software program (Biosoft Company, Cambridge, UK). 2.5. ATPases Activity Pgp, MRP1, and BCRP had been immunoprecipitated from 100 g of membrane-associated protein, then the price of ATP hydrolysis, an index from the catalytic routine and a required stage for substrate efflux, was assessed spectrophotometrically [27]. In each group of tests, 0.5 mM Na3VO4 was included in the reaction mix to measure the Na3VO4-sensitive rate of ATP hydrolysis. Results were indicated as nmoles hydrolyzed phosphate/mg protein. 2.6. Caspase 3 Activity Cells were lysed in 0.5 mL of lysis buffer (20 mM Hepes/KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiotreitol DTT, 1 mM PMSF, 10 g/mL leupeptin, pH 7.5). Twenty micrograms of cell lysates was incubated for 1 h at 37 C with 20 M of the fluorogenic substrate of caspase-3 Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), in 0.25 mL of assay buffer (25 mM Hepes, 0.1% 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate CHAPS, 10% sucrose, and 10 mM DTT, 0.01% egg albumin, pH 7.5). The reaction was stopped by adding 0.75 mL of ice-cold Ethisterone 0.1% trichloroacetic acid, and the fluorescence of AMC fragment released by active caspases was go through using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Devices, Winooski, VT, USA). Excitation and emission wavelengths were 380 and 460 nm, respectively. Fluorescence was converted in nmoles AMC/mg cellular proteins, using a calibration curve prepared previously with standard solutions of AMC. 2.7. Cell Viability Cell viability was evaluated using the ATPLite kit (PerkinElmer, Waltham, MA, USA). The results were indicated as percentage of viable cells in each experimental condition versus untreated cells (regarded as 100% viable). 2.8. Proximity Ligation Assay The CRTCPgp connection was measured with the DuoLink In Situ Kit (Sigma-Merck), as per manufacturers instructions, using the mouse anti-Pgp (UIC-2, Millipore; dilution 1/50) and the rabbit anti-CRT (PA3-900, ABR-Affinity BioReagents Inc.; dilution 1/50) antibodies. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Cells were examined using a Leica DC100 fluorescence microscope (Leica Microsystem, Wetzlar, Germany). 2.9. Confocal Microscope Analysis Cells.
Categories