Antibodies against p21 (sc-471) and Bax (sc-493) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Noxa antibody (ab13654) was purchased from Abcam (Cambridge, UK). p53 antibody (1C12) was purchased from Cell Signaling (Danvers, MA, USA). β-Actin antibody (A5441) was purchased from Sigma Aldrich. Caspase-3 antibody (CS-9665S) and cleaved caspase-3 (Cleaved Caspase-3 (CS-9661S)) were purchased from Cell Signaling and generously provided by Dr. Jun Tsuji (NCSU).
Sc 493
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Sc-493 is a laboratory reagent manufactured by Santa Cruz Biotechnology. It is used as a research tool in scientific investigations. The core function of Sc-493 is to serve as a control or reference material in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg fitc ab6717 1, by Abcam (3 mentions)
Nb100 56113, by Novus Biologicals (3 mentions)
Sc 783, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Sc 7382, by Santa Cruz Biotechnology (2 mentions)
Sc 492, by Santa Cruz Biotechnology (2 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (2 mentions)
β actin antibody a5441, by Merck Group (1 mentions)
P53 antibody 1c12, by Cell Signaling Technology (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Sc 393031, by Santa Cruz Biotechnology (1 mentions)
Sc 2010, by Santa Cruz Biotechnology (1 mentions)
Sc 56076, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Sc 6243, by Santa Cruz Biotechnology (1 mentions)
Sc 6246, by Santa Cruz Biotechnology (1 mentions)
Sepharose cl 2b, by GE Healthcare (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
15 protocols using sc 493
1
Antibody Sourcing for Apoptosis Assay
2
Protein Expression Profiling in Treated Cells
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Cells were cultured in 3.4 cm2-diameter culture dishes over sterile cover slips until 80% confluence and treated with C1 and C2 (IC50 of C1: 4.69; C2: 8.36 μg/mL) for 24 h. After the treatment, the cells were fixed in 4% formaldehyde for 15 min at room temperature and washed twice with 1x PBS, followed by incubating the cells with permeabilization buffer (0.01% v/v Triton X-100) for 15 min and then washed twice with 1x PBS. Cells were blocked in PBS containing 3% bovine serum albumin for 1 h, and primary antibody recognizing p53 (Santa Cruz Biotechnology, SC393031), Bax (Santa Cruz Biotechnology, SC493), and caspase 9 (Santa Cruz Biotechnology, SC56076) were added at 1 : 100 dilution for 2 h at room temperature. The cells were washed twice with 1x PBS and FITC (Santa Cruz Biotechnology, SC2010; SC2359) conjugated secondary antibodies were added and incubated for 2 h. The nucleus was stained by DAPI and the slides were examined using the Carl Zeiss Axio Observer Z1 with the filter set 358Ex/461Em.
3
Protein Expression Analysis Protocol
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Protein levels was determined as previously described [36 (link)] with antibodies against p53 (SC-6243, 1:500, Santa Cruz), p-p53ser15 (9284S 1:500, Cell Signal), p21 (SC-6246, 1:1000, Santa Cruz), BAX (SC-493, 1:500, Santa Cruz), c-PARP (9542 S 1:500, Cell Signal), and β-actin (2228, 1:20000, Sigma Aldrich).
4
BAX-Mediated Liposome Release Assay
5
Immunofluorescence Analysis of Apoptosis Markers
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The expression of Bcl-2, Bax and cleaved caspase-3 proteins were investigated by immunofluorescence method as previously described [54 (link)]. In brief, the 4T1 cells were seeded in a 6-well plate and exposed to the DE-EDCP and cisplatin at concentration of 31.25 μM for 24 hours. After washing the cells twice with PBS, they were fixed in 4% paraformaldehyde at 25°C for 20 min. The cells were stained with rabbit polyclonal antibody specific for Bcl-2 (sc-783, Santa Cruz Biotech. Inc CA, USA), Bax (sc-493, Santa Cruz Biotech. Inc CA, USA) and active/cleaved caspase-3 (NB100-56113, Novus Biologicals, UK). After incubation, the cells were washed and treated with appropriate secondary antibody, goat anti-rabbit IgG FITC (Ab6717-1, Abcam, Cambridge, United Kingdom). The sections were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and analyzed at x 200 magnification using fluorescent microscope (Olympus BX 51).
6
Western Blot Analysis of Apoptosis Markers
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RIPA buffer along with the phenylmethanesulfonyl fluoride (Beyotime, Nanjing, China) inhibitor was used to extract proteins from kidney tissues and NRK-52E cells. The protein concentration was determined using a BCA protein assay kit (Beyotime, Nanjing, China). 10% SDS-PAGE gels were used and protein samples with a concentration of 40 µg were loaded into each well of the gel. Nitrocellulose membranes (Millipore, Billerica, USA) were used to transfer proteins. Once the transfer was complete, the membranes were incubated for blocking using 5% skim milk made in TBST buffer for 2 h. Next, they were incubated overnight with various primary antibodies at 4 °C such as Bax (1:500 dilutions; sc-493; Santa Cruz Biotechnology), Bcl-2 (1:1000; sc-7382; Santa Cruz Biotechnology) and caspase-3 (1:1000; sc7148; Santa Cruz Biotechnology). Next day, they were incubated at room temperature for 1.5 h with the secondary antibody coupled with horseradish peroxidase (1:1000, Santa Cruz Biotechnology, CA, USA). The protein was detected using ECL reagents (Millipore, Billerica, USA).
7
Western Blot Analysis of Bax and Bcl-2
8
Protein Expression Profiling in Cell Lines
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The cells were placed in the radioimmune precipitation assay buffer and then the the cellular proteins were separated with SDS-15% polyacrylamide gel electrophoresis under denaturing conditions. Then, after blocked with 5% skim milk, the nitrocellulose membrane transferred with proteins were incubated with primary antibodies against HVEM (1:500; Ab62462, Abcam, USA), HIF-1 α (1:500; Ab113642, Abcam), Bcl-2 (1:400; Sc-492, Santa Cruz Biotechnology), Bax (1:300; Sc-493, Santa Cruz Biotechnology), AKT [1:1000; #9272, Cell Signaling Technology (CST)], p-AKT (1:1000; #9271, CST), mTOR (1:1000; #2972, CST), p-mTOR (1:1000; #2971; CST) and GAPDH antibodies (1:2000; #5174, CST, Danvers, MA, USA), followed by a horseradish-peroxidase-conjugated secondary antibody (Beyotime, Shanghai, China). The signals were determined by an enhanced chemiluminescence system (Bio- Rad). GAPDH was selected as the internal control.
9
Pt(S-pr-thiosal)2 Induced Cell Apoptosis
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BCL1 cells were seeded in a six-well plate and exposed to the Pt(S-pr-thiosal)2 (0.05 mg/mL) for 12 h. Cells were washed twice with phosphate-buffered saline and then fixed in 4% paraformaldehyde at 25 °C for 20 min. For cell staining, rabbit polyclonal antibody specific for Bcl-2 (sc-783, Santa Cruz Biotech Inc., Santa Cruz, CA, USA), Bax (sc-493, Santa Cruz Biotech Inc.), and phospho NF-κB (ab131109 Abcam, Cambridge, UK) and active/cleaved caspase 3 (NB100-56113, Novus Biologicals) were used. After incubation, the cells were washed and treated with appropriate secondary antibody, goat anti-rabbit IgG FITC (Ab6717-1, Abcam). The sections were mounted with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). For the cell analysis, fluorescent microscope (Olympus BX 51) was used at 200× magnification.
10
Western Blot Analysis of Alzheimer's Biomarkers
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The protein expression of SIRT2, bcl-2, and bax was assayed by western blotting. Briefly, the cerebral cortex, cerebellum, and hippocampus tissue lysates were prepared on ice with 300 μl of RIPA Lysis Buffer per 20 mg of tissue (Santa Cruz Biotechnology, TX). The samples containing 20 µg of total protein were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% or 10%). The protein bands were then transferred to nitrocellulose membranes (Bio-Rad, CA) blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% (w/v) non-fat dry milk (Santa Cruz Biotechnology, TX) for overnight at +4 °C. Following the transfer of the proteins, the membranes were incubated with primary antibodies against SIRT2 (1:500, sc-28298; Santa Cruz Biotechnology, TX), bcl-2 (1:1000, MA1-12246), bax (1:500, sc-493), or β-actin (1:125, sc-130657; Santa Cruz Biotechnology, TX) for 2 hours. Then the membranes were incubated for 1 hour with horseradish peroxidase (HRP) conjugated secondary antibodies (anti-rabbit, 1:5000, sc-2004; or anti-mouse, 1:5000, sc-2005; Santa Cruz Biotechnology, TX) in room temperature. Enhanced chemiluminescence detection reagent (Pierce™ Thermo Sci, IL) was used to visualize the specific protein bands on X-ray film (Carestream Health Inc. NY). The bands were quantified by using ImageJ software.
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