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Sc 397

Manufactured by Santa Cruz Biotechnology
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Sc-397 is a laboratory instrument designed for cell culture and biochemical applications. It is a centrifuge that separates different components of a liquid mixture based on their density and size. The Sc-397 operates at variable speeds to accommodate a range of sample types and volumes.

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Lab products found in correlation

Sc 6243, by Santa Cruz Biotechnology (5 mentions) Ab1791, by Abcam (3 mentions) A5441, by Merck Group (2 mentions) T7 p53dd pcdna3, by Addgene (2 mentions) Powerplex16hs assay, by Promega (2 mentions) Dmem f12, by Merck Group (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Sc 1641, by Santa Cruz Biotechnology (2 mentions) Sc 81178, by Santa Cruz Biotechnology (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Ab97959, by Abcam (1 mentions) Sc 365964, by Santa Cruz Biotechnology (1 mentions) Supersignal west pico chemiluminescent substrate, by Merck Group (1 mentions) Ab2769, by Abcam (1 mentions) Ab86430, by Abcam (1 mentions) Anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions) Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Bca kit, by Beyotime (1 mentions)

37 protocols using sc 397

1

Western Blot Analysis of Cell Lysates

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Tissues were dissected, flash frozen in liquid nitrogen, and homogenized in RIPA buffer containing protease inhibitors. Total protein from cell lysates was prepared as previously described [38 (link)]. Total protein was separated by SDS-PAGE then transferred to a PVDF membrane (Millipore, IPFL00010). Primary antibodies for human cells were anti-p53 (mouse monoclonal, OP43 Calbiochem, 1/1000), anti-p21 (rabbit polyclonal, sc-397 Santa-Cruz, 1/1000), anti-RB (mouse monoclonal, 9309 Cell Signaling, 1/1000), anti-pRB-Ser780 (rabbit monoclonal, 8180 Cell Signaling, 1/1000), anti-Actin (rabbit polyclonal, A2066 Sigma, 1/10000), and anti-Tubulin (rabbit polyclonal, 926–42211 Licor, 1/5000). Primary antibodies for mouse tissues were p53 (mouse monoclonal, 2524 Cell Signaling, 1/1000) and p21 (rabbit polyclonal, sc-397 Santa-Cruz, 1/1000). Secondary antibodies were conjugated to Alexa Fluor 680 (Life Technology) or IRDye 800 (LiCOR) for scanning in the LiCOR Odyssey system.
Saison-Ridinger M., DelGiorno K.E., Zhang T., Kraus A., French R., Jaquish D., Tsui C., Erikson G., Spike B.T., Shokhirev M.N., Liddle C., Yu R.T., Downes M., Evans R.M., Saghatelian A., Lowy A.M, & Wahl G.M. (2017). Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy. PLoS ONE, 12(12), e0189051.
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2

Western Blot Analysis of Protein Markers

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Cells and tumor tissues were lysed with RIPA Lysis buffer (Beyotime, Shanghai, China), and protein concentrations were determined by the BCA kit (Beyotime, Shanghai, China). Each sample was separated by 8-12% SDS-PAGE, blocked with 5% BSA (Albumin from bovine serum) for 2 h at room temperature, and incubated with primary antibodies to β-tubulin, Sox2 (#14962, 1:100, CST, MA, USA; sc-365964, 1:100, Santa Cruz, TX, USA; ab97959, 1:1000, Abcam, Cambridge, UK), AhR (ab2769, 1:500, Abcam, Cambridge, UK), STAT3 (#12640, 1:1000, CST, MA, USA), Y-STAT3 (ab76315, 1:2000, Abcam, Cambridge, UK), S-STAT3 (ab86430, 1:250, Abcam, Cambridge, UK), PCNA (#2586, 1:2000, CST, MA, USA), IDO1 (#51851, 1:500, CST, MA, USA), p53 (#2524, 1:1000, CST, MA, USA), p27 (#3686, 1:1000, CST, MA, USA), and p21 (sc-397, 1:200, Santa Cruz, TX, USA) overnight at 4 oC. Primary antibodies were detected with a goat anti-rabbit IgG-HRP (sc-2004, 1:2000, Santa Cruz, TX, USA) or anti-mouse IgG-HRP (sc-2005, 1:2000, Santa Cruz, TX, USA). The blots were developed using Super Signal West Pico chemiluminescent substrate (Millipore, Billerica, MA, USA).
Jia Q., Yang F., Huang W., Zhang Y., Bao B., Li K., Wei F., Zhang C, & Jia H. (2019). Low Levels of Sox2 are required for Melanoma Tumor-Repopulating Cell Dormancy. Theranostics, 9(2), 424-435.
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3

Embryonic Development Analysis

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Embryos were collected from timed pregnancy at various stages of gestation for comparison. The size of the embryos was determined based on the number of pixels in the embryos calculated using software ImageJ. For histological analysis, embryos were fixed in formalin overnight. Serial 5-µm sagittal sections were collected and hematoxylin and eosin staining was performed according to standard procedures. The sections were also immunostained using antibodies against SET (bethyl, A302-261A), p53 (Leica, CM5), and Cleaved Caspase 3 (Cell Signaling, 9661), p21 (Santa Cruz, SC397), and counter-stained with hematoxylin.
Kon N., Wang D, & Gu W. (2019). Loss of SET reveals both the p53-dependent and the p53-independent functions in vivo. Cell Death & Disease, 10(3), 237.
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4

Whole Cell Lysate Preparation and Immunoblotting

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Whole cell lysates were prepared in RIPA lysis buffer supplemented with protease inhibitor cocktail (Complete Mini; Roche, Basel, Switzerland). The lysates were sonicated for 5 minutes, centrifuged at 12 000 × g for 20 minutes at 4°C, and the supernatants were collected. Immunoblotting was carried out as previously described.29 The following primary antibodies were used at the indicated dilutions: anti‐β‐actin (1:10 000, A5441; Sigma‐Aldrich), anti‐p21 (1:1000, Sc‐397; Santa Cruz Biotechnology, Dallas, TX, USA), anti‐cleaved poly ADP‐ribose polymerase (PARP, 1:1000, #9541; Cell Signaling Technology, Danvers, MA, USA), anti‐IDH1 (1:200, 014‐24061; Wako), and anti‐IDH1‐R132S (1:200, 015‐24091; Wako).
Fujiwara H., Tateishi K., Kato H., Nakatsuka T., Yamamoto K., Tanaka Y., Ijichi H., Takahara N., Mizuno S., Kogure H., Matsubara S., Nakai Y, & Koike K. (2018). Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma to the BET inhibitor JQ1. Cancer Science, 109(11), 3602-3610.
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5

HT-29 Cell Protein Expression Analysis

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HT-29 cells were seeded in 100-mm dishes and treated at 50% confluency according to indicated treatment conditions, then harvested by trypsinization at 8 h after hypericin activation. Western blot analyses were conducted using total cellular protein extracts (30–50 mg). The blots were incubated overnight at +4 °C with the following specific primary antibodies: anti-HDAC1 (1/1000, ab 46985, Abcam), anti-HDAC3 (1/500, sc-11417, Santa Cruz Biotechnology), anti-HDAC6 (1/500, sc-11420, Santa Cruz Biotechnology), anti-CDKN1A (1/500, sc-397, Santa Cruz Biotechnology), anti-H3 (1:2500, ab 1791, Abcam), anti-H3ac (1:2500, MILL 17-245, Merck Millipore), followed by incubation with species-matched secondary antibodies. β-Actin (1:5000, A5441, Sigma-Aldrich) or H3 was used as the loading control. Specific proteins were detected by exposing membranes to ChemiDoc XRS+ System (Bio-Rad Laboratories) after incubation with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Densitometry analysis was performed using ImageJ software.
Halaburková A., Jendželovský R., Kovaľ J., Herceg Z., Fedoročko P, & Ghantous A. (2017). Histone deacetylase inhibitors potentiate photodynamic therapy in colon cancer cells marked by chromatin-mediated epigenetic regulation of CDKN1A. Clinical Epigenetics, 9, 62.
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6

Western Blot Analysis of Cell Signaling Proteins

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Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay reagent (Bio-Rad) according to manufacturer instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-p21 (1:500; sc-397), anti-IκBα (1:500; sc-371), anti-p53 (1:500; sc-126, all from Santa Cruz Biotechnology), anti-p-p53 (1:500; 9286, Cell Signaling Technology), anti-ID1 (1:2,000; BCH-1-195-14, Biocheck, Foster City, CA, USA), anti-ID2 (1:500, sc-489, Santa Cruz Biotechnology), anti-ID3 (1:500, sc-490, Santa Cruz Biotechnology), anti-ID4 (1:200; ab49261, Abcam), and anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with a horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).
Jeon H.Y., Ham S.W., Kim J.K., Jin X., Lee S.Y., Shin Y.J., Choi C.Y., Sa J.K., Kim S.H., Chun T., Jin X., Nam D.H, & Kim H. (2019). Ly6G+ inflammatory cells enable the conversion of cancer cells to cancer stem cells in an irradiated glioblastoma model. Cell Death and Differentiation, 26(10), 2139-2156.
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7

Generating p53-Deficient Ovarian and Fallopian Cell Lines

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We have previously reported the generation of the IOE11 TERT-immortalized ovarian surface epithelial cell line23 (link). IOE11 cultured in NOSE-CM52 (link). To generate a p53-deficient line, IOE11 cells were transfected with T7-p53DD-pcDNA3 (Addgene plasmid number 25989) and positive clones (IOE11-DNp53) selected with 125 μg ml−1 G418. Loss of p53 function was confirmed by irradiating IOE11-DNp53 and control cells with 6 Gy ionizing radiation and immunoblotting cell lysates for p21 expression (sc-397, 1:1,000 dilution, Santa Cruz Biotechnology) 24 h later. Immortalized fallopian tube secretory epithelial cell lines (FT33-shp53-R24C and FT246-shp53-R24C) have been previously described22 (link) and were cultured in DMEM/F12 (Sigma) supplemented with 2% Ultroser G (Crescent Chemicals) or 10% fetal bovine serum (FBS; Hyclone, Thermo Fisher). For 3C, HEY cells were grown in RPMI containing 10% FBS and OVCA429 cells were cultured in EMEM supplemented with 10% FBS, 1 × non-essential amino acids and 1 × sodium pyruvate. All cell lines used in this study were routinely tested for Mycoplasma infection using a Mycoplasma-specific PCR, and, for cell line authentication, short tandem repeats profiled using the PowerPlex16HS Assay (Promega, University of Arizona Genetics Core).
Lawrenson K., Li Q., Kar S., Seo J.H., Tyrer J., Spindler T.J., Lee J., Chen Y., Karst A., Drapkin R., Aben K.K., Anton-Culver H., Antonenkova N., Baker H., Bandera E.V., Bean Y., Beckmann M.W., Berchuck A., Bisogna M., Bjorge L., Bogdanova N., Brinton L.A., Brooks-Wilson A., Bruinsma F., Butzow R., Campbell I.G., Carty K., Chang-Claude J., Chenevix-Trench G., Chen A., Chen Z., Cook L.S., Cramer D.W., Cunningham J.M., Cybulski C., Dansonka-Mieszkowska A., Dennis J., Dicks E., Doherty J.A., Dörk T., du Bois A., Dürst M., Eccles D., Easton D.T., Edwards R.P., Eilber U., Ekici A.B., Fasching P.A., Fridley B.L., Gao Y.T., Gentry-Maharaj A., Giles G.G., Glasspool R., Goode E.L., Goodman M.T., Grownwald J., Harrington P., Harter P., Hasmad H.N., Hein A., Heitz F., Hildebrandt M.A., Hillemanns P., Hogdall E., Hogdall C., Hosono S., Iversen E.S., Jakubowska A., James P., Jensen A., Ji B.T., Karlan B.Y., Kruger Kjaer S., Kelemen L.E., Kellar M., Kelley J.L., Kiemeney L.A., Krakstad C., Kupryjanczyk J., Lambrechts D., Lambrechts S., Le N.D., Lee A.W., Lele S., Leminen A., Lester J., Levine D.A., Liang D., Lissowska J., Lu K., Lubinski J., Lundvall L., Massuger L.F., Matsuo K., McGuire V., McLaughlin J.R., Nevanlinna H., McNeish I., Menon U., Modugno F., Moysich K.B., Narod S.A., Nedergaard L., Ness R.B., Azmi M.A., Odunsi K., Olson S.H., Orlow I., Orsulic S., Weber R.P., Pearce C.L., Pejovic T., Pelttari L.M., Permuth-Wey J., Phelan C.M., Pike M.C., Poole E.M., Ramus S.J., Risch H.A., Rosen B., Rossing M.A., Rothstein J.H., Rudolph A., Runnebaum I.B., Rzepecka I.K., Salvesen H.B., Schildkraut J.M., Schwaab I., Sellers T.A., Shu X.O., Shvetsov Y.B., Siddiqui N., Sieh W., Song H., Southey M.C., Sucheston L., Tangen I.L., Teo S.H., Terry K.L., Thompson P.J., Timorek A., Tsai Y.Y., Tworoger S.S., van Altena A.M., Van Nieuwenhuysen E., Vergote I., Vierkant R.A., Wang-Gohrke S., Walsh C., Wentzensen N., Whittemore A.S., Wicklund K.G., Wilkens L.R., Woo Y.L., Wu X., Wu A.H., Yang H., Zheng W., Ziogas A., Monteiro A., Pharoah P.D., Gayther S.A, & Freedman M.L. (2015). Cis-eQTL analysis and functional validation of candidate susceptibility genes for high-grade serous ovarian cancer. Nature Communications, 6, 8234.
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8

Detecting Smad Signaling Pathway

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Antibodies specific for Smad3 (#9523), p-Smad3 (Ser423/425, #9520), Smad2 (#3103), and p-Smad2 (Ser465/467, #3101) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies specific for p-Smad2/3 (sc-11769), Ski (sc-9140), p21 (sc-397), and actin (sc-1616) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-conjugated anti-rabbit (specific for Smad3, p-Smad3, p-Smad2, Ski, p21, and actin) or anti-mouse (specific for Smad2) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-rabbit FITC (ZF-0311), anti-mouse TRITC (ZF-0313), and anti-goat TRITC (ZF-0317) were purchased from ZhongShan Golden Bridge Biotechnology (Beijing, China).
Li P., Wang Q.S., Zhai Y., Xiong R.P., Chen X., Liu P., Peng Y., Zhao Y., Ning Y.L., Yang N, & Zhou Y.G. (2020). Ski mediates TGF-β1-induced fibrosarcoma cell proliferation and promotes tumor growth. Journal of Cancer, 11(20), 5929-5940.
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9

Senescence Markers Evaluation in MSCs

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MSCs senescence was evaluated by the expression of the DNA damage marker H2AX (1:200; Abcam, Cambridge, UK) using Western blot, while SA-ß-GAL activity of MSCs was measured using a Cellular Senescence Activity Assay kit (Enzo Life Sciences, Farmingdale, NY, USA) following the vendor’s protocol. p21 and p16 immunofluorescent staining was performed following standard protocols20 (link), using primary antibodies: p16 (ab118459), mouse monoclonal (cell line JC8), Santa Cruz (SC-56330, Santa Cruz, CA, USA); p21, rabbit polyclonal (c19), Santa Cruz (SC-397). The percentage of the positively stained area was quantified using ZEN® 2012 blue edition (ZEISS, Munich, Germany).
Meng Y., Eirin A., Zhu X.Y., Tang H., Hickson L.J., Lerman A., van Wijnen A.J, & Lerman L.O. (2018). Micro-RNAS Regulate Metabolic Syndrome-induced Senescence in Porcine Adipose Tissue-derived Mesenchymal Stem Cells through the P16/MAPK Pathway. Cell Transplantation, 27(10), 1495-1503.
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10

Protein Extraction and Western Blot Analysis

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Whole cell extracts were obtained by washing cells in phosphate-buffered saline (PBS) before lysis in SDS loading buffer (2% SDS, 10% (v/v) glycerol, 25 mM TCEP and 62.5 mM Tris-HCl, pH 6.8). Extracts were heated at 95 °C for 5 min, followed by shearing with 10 strokes through a 25G needle. Protein concentrations were determined by Bradford assay (Bio-Rad) or NanoDrop (Thermo Scientific). SDS-PAGE and western blotting were performed using the Novex NuPAGE SDS-PAGE gel system (Life Technologies) or the SE400 and TE42 systems from Hoefer. The following antibodies were used at the indicated dilutions: CHK1 (sc8408, Santa Cruz Biotechnology, 1/1000), CHK1-pS345 (2348, Cell Signaling Technology, 1/10,000), CHK2-pT68 (2661, Cell Signaling Technology, 1/500), DNA-PKcs (MS-369-P1, Thermo Scientific, 1/200), GFP (11814460001, Roche, 1/5000), γH2AX (05-636, Millipore, 1/1000), H3 (ab1791, Abcam, 1/50,000), H3-pS10 (ab14955, Abcam, 1/5000), Ku70 (ab3114, Abcam, 1/1000), Ku80 (MS-285-P1, Thermo Scientific, 1/2000), p21 (sc397, Santa Cruz Biotechnology, 1/1000), p53 (554293, BD Biosciences, 1/6000), PAXX (ab126353, Abcam, 1/500), RPA1, (A300-241A, Bethyl Laboratories, 1/1000), TopBP1 (A300-111A, Bethyl Laboratories, 1/5000), XLF (ab33499, Abcam, 1/650) and XRCC4 (ab145, Abcam, 1/3000).
Ochi T., Blackford A.N., Coates J., Jhujh S., Mehmood S., Tamura N., Travers J., Wu Q., Draviam V.M., Robinson C.V., Blundell T.L, & Jackson S.P. (2015). PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair. Science (New York, N.Y.), 347(6218), 185-188.
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