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FUJIFILM leupeptin
Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with <t>leupeptin,</t> the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P
Leupeptin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "?-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor"

Article Title: ?-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093509

Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P
Figure Legend Snippet: Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P

Techniques Used: Transfection, Incubation, Software, Labeling

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Sonication:

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Flow Cytometry:

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Article Snippet: .. The following antibodies and reagents were obtained from commercial sources: rabbit anti-GFP (598) and rat antihemagglutinin (anti-HA) (561; Medical and Biological Laboratories Co. Ltd., Nagoya, Japan); mouse anti-β-actin (A1978), pepstatin A (P5318), doxorubicin (D-1515), and Hoechst 33342 (Sigma-Aldrich, St. Louis, MO); anti-DYKDDDDK tag (014-22383), anti-DYKDDDDK tag beads (016-22784), anti-V5 tag beads (016-24381), Phos-tag acrylamide, and leupeptin (334-40414) (Wako Pure Chemical Industries, Osaka, Japan); MG-132 (Nakalai Tesque, Tokyo, Japan); VP-16 (E0675) (Tokyo Medical Industry, Tokyo, Japan); lambda phosphatase (sc-200312; Santa Cruz Biotechnology, Dallas, TX); Picagene (Toyo Ink); rabbit polyclonal anti-V5 tag (PM003; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan); rabbit anti-RASSF6 (11921-1-AP; Proteintech, Rosemond, IL); protein G-Sepharose 4 Fast Flow (GE Healthcare, Little Chalfont, United Kingdom); mouse anti-cytochrome c (6H2 B4) (556432), mouse anti-human Rb (554136), and mouse anti-underphosphorylated-Rb (554164) (BD Biosciences, San Jose, CA); mouse anti-Myc (9E10; American Type Culture Collection, Manassas, VA); rabbit polyclonal anti-phospho-Rb (Ser608) (2181) and rabbit polyclonal anti-phospho-Rb (Ser807/811) (9308) (Cell Signaling Technology, Danvers, MA); mouse monoclonal anti-Bmi-1 (05-637) and anti-phospho-histone H2A.X (Ser139) (JBW341) (Merck, Kenilworth, NJ); and rabbit polyclonal anti-phospho-Rb (Thr821) (44-582G; Thermo Fisher Scientific, Waltham, MA). .. HEK293FT, HCT116, HCT116 p53−/− , and HeLa cells were cultured in Dulbecco's modified Eagle medium containing 10% (vol/vol) fetal bovine serum and 10 mM HEPES-NaOH, pH 7.4, under 5% CO2 at 37°C.

Western Blot:

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    FUJIFILM leupeptin showed prp levels 1 4
    Leupeptin Showed Prp Levels 1 4, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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