ON-TARGETplus Human AHR (196) siRNA - Individual

Protocol tips

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	The siRNA knockdowns were performed as detailed in our prior reports [33, 34]. Briefly, 200,000 cells (MCF-7 or MDA-MB-231) in 1 mL of DMEM supplemented with 10% FBS were mixed directly with 100 nM of siRNA that was non-targeting, AHR-targeting or LAT1-targeting and 3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and immediately plated in 35 mm tissue culture plates for 48 hrs. MCF-7 cells were then treated with vehicle (DMSO) or 10 nM TCDD for 16 hrs. Treatments were removed and cells were rinsed once with PBS. For Western blotting, total protein was extracted by scraping cells in 2× Laemmli Sample Buffer containing β-mercaptoethanol (BME). Laemmli sample buffer and BME were purchased from BIO-RAD. Standard Western blotting techniques were used to analyze ∼ 10 μg of protein per sample (please refer to our prior reports for technical details [33, 34]). Western blot analysis of GAPDH was used to confirm equal protein loading. Blots were probed with anti-GAPDH antibody (diluted 1:10,000), anti-AHR antibody (diluted 1:5,000) or anti-LAT1 antibody (diluted 1:2,000) overnight at 4oC, followed by incubation with anti-HRP secondary antibody (1:5000) for 1 hr at room temperature. The blots were then rinsed with PBS + 0.1% tween 20, and then developed with enhanced chemiluminescent substrate Millipore Corp., (Billerica, MA). The anti-GAPDH antibody was purchased from Millipore (Cat #MAB374). The anti-AHR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat #H-211) and the anti- LAT1 antibody was purchased from Cell Signaling Technology (Danvers, MA, Cat #5347). Densitometry was calculated with ImageJ PC-based software (National Institute of Health). The Student-Newman–Keuls (SNK) post-hoc test was used to determine statistically significant differences among groups following one-way analysis of variance (ANOVA).

Protocol tips

The siRNA knockdowns were performed as detailed in our prior reports [33, 34]. Briefly, 200,000 cells (MCF-7 or MDA-MB-231) in 1 mL of DMEM supplemented with 10% FBS were mixed directly with 100 nM of siRNA that was non-targeting, AHR-targeting or LAT1-targeting and 3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and immediately plated in 35 mm tissue culture plates for 48 hrs. MCF-7 cells were then treated with vehicle (DMSO) or 10 nM TCDD for 16 hrs. Treatments were removed and cells were rinsed once with PBS. For Western blotting, total protein was extracted by scraping cells in 2× Laemmli Sample Buffer containing β-mercaptoethanol (BME). Laemmli sample buffer and BME were purchased from BIO-RAD. Standard Western blotting techniques were used to analyze ∼ 10 μg of protein per sample (please refer to our prior reports for technical details [33, 34]). Western blot analysis of GAPDH was used to confirm equal protein loading. Blots were probed with anti-GAPDH antibody (diluted 1:10,000), anti-AHR antibody (diluted 1:5,000) or anti-LAT1 antibody (diluted 1:2,000) overnight at 4oC, followed by incubation with anti-HRP secondary antibody (1:5000) for 1 hr at room temperature. The blots were then rinsed with PBS + 0.1% tween 20, and then developed with enhanced chemiluminescent substrate Millipore Corp., (Billerica, MA). The anti-GAPDH antibody was purchased from Millipore (Cat #MAB374). The anti-AHR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat #H-211) and the anti- LAT1 antibody was purchased from Cell Signaling Technology (Danvers, MA, Cat #5347). Densitometry was calculated with ImageJ PC-based software (National Institute of Health). The Student-Newman–Keuls (SNK) post-hoc test was used to determine statistically significant differences among groups following one-way analysis of variance (ANOVA).

Publication protocol

The siRNA knockdowns were performed as detailed in our prior reports [33, 34]. Briefly, 200,000 cells (MCF-7 or MDA-MB-231) in 1 mL of DMEM supplemented with 10% FBS were mixed directly with 100 nM of siRNA that was non-targeting, AHR-targeting or LAT1-targeting and 3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and immediately plated in 35 mm tissue culture plates for 48 hrs. MCF-7 cells were then treated with vehicle (DMSO) or 10 nM TCDD for 16 hrs. Treatments were removed and cells were rinsed once with PBS. For Western blotting, total protein was extracted by scraping cells in 2× Laemmli Sample Buffer containing β-mercaptoethanol (BME). Laemmli sample buffer and BME were purchased from BIO-RAD. Standard Western blotting techniques were used to analyze ∼ 10 μg of protein per sample (please refer to our prior reports for technical details [33, 34]). Western blot analysis of GAPDH was used to confirm equal protein loading. Blots were probed with anti-GAPDH antibody (diluted 1:10,000), anti-AHR antibody (diluted 1:5,000) or anti-LAT1 antibody (diluted 1:2,000) overnight at 4oC, followed by incubation with anti-HRP secondary antibody (1:5000) for 1 hr at room temperature. The blots were then rinsed with PBS + 0.1% tween 20, and then developed with enhanced chemiluminescent substrate Millipore Corp., (Billerica, MA). The anti-GAPDH antibody was purchased from Millipore (Cat #MAB374). The anti-AHR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat #H-211) and the anti- LAT1 antibody was purchased from Cell Signaling Technology (Danvers, MA, Cat #5347). Densitometry was calculated with ImageJ PC-based software (National Institute of Health). The Student-Newman–Keuls (SNK) post-hoc test was used to determine statistically significant differences among groups following one-way analysis of variance (ANOVA).

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