Sc 5546

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-5546 is a laboratory product from Santa Cruz Biotechnology. It is a tool for scientific research, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information is not available.

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Lab products found in correlation

Ab48394, by Abcam (1 mentions) C2206, by Merck Group (1 mentions) A g plus agarose beads, by Santa Cruz Biotechnology (1 mentions) Sc 2025, by Abcam (1 mentions) Sc 1008, by Santa Cruz Biotechnology (1 mentions) E cadherin, by GeneTex (1 mentions) Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) 12 well dishes, by Corning (1 mentions) Amersham ecl anti mouse na 931, by Santa Cruz Biotechnology (1 mentions) Anti goat sc 2020, by Santa Cruz Biotechnology (1 mentions) Sc 40, by Abcam (1 mentions) F1804, by Merck Group (1 mentions) α tubulin, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Rpn2232, by Cytiva (1 mentions) L8918, by Merck Group (1 mentions) Polyvinylidene difluoride membrane, by Roche (1 mentions) Phospho mtor, by Abcam (1 mentions) P ulk1, by Cell Signaling Technology (1 mentions) P ulk1s757, by Cell Signaling Technology (1 mentions)

8 protocols using sc 5546

Protein Interaction Analysis by Co-Immunoprecipitation

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SDS-PAGE and immunoblots were performed as previously described(20 (link)) using antibodies for β-catenin (C2206; Sigma, St. Louis, MO), tubulin (sc-5546; Santa Cruz Biotechnology, Santa Cruz, CA), VDR (sc-13133), E-cadherin (sc-21791), DKK-1 (GTX62902; GeneTex, Irvine, CA), LRP6 (CST3395; Cell Signaling Technology, Danvers, MA), RXRα (CST3085), RXRβ (CST8715), LDLRAP1 (C20125; LSBio, Seattle, WA) or LC3B (ab48394; Abcam, Cambridge, MA). For LRP6 and LC3B immunoblots, lysates were resolved on 4-20% gradient gels. Co-immunoprecipitations were performed as previously described(21 (link)). Briefly, AsPC-1 lysates were pre-cleared with A/G-PLUS agarose beads (Santa Cruz; sc-2003) and immunoprecipitated using antibody to β-catenin (BD Transduction Laboratories; 610153), VDR (Santa Cruz; sc-1008) or control isotype-matched IgG (Santa Cruz; sc-2025 or Abcam ab46540). After multiple washes, immune complexes were boiled in 6× SDS-load dye, resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting.

Arensman M.D., Nguyen P., Kershaw K.M., Lay A.R., Ostertag-Hill C.A., Sherman M.H., Downes M., Liddle C., Evans R.M, & Dawson D.W. (2015). Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer. Molecular cancer research : MCR, 13(11), 1509-1519.

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Exosome Immunophenotyping via Bead Capture

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Tumor exosomes (10 µg protein) in 0.5 mL PBS were mixed overnight at 4°C with 5 µL of 4 µM aldehyde-activate latex beads (4% w/v) (Invitrogen). The beads were then blocked by adding 0.5 mL of 1% bovine serum albumin (BSA) for 1 hr followed by 0.1 mL of 100 mM glycine for an additional hour. The beads were then washed and resuspended in PBS. Antibodies included rabbit anti-alix (sc-99010; Santa Cruz) and rabbit anti-tubulin (sc-5546; Santa Cruz) as an isotype control followed by CY3-labeled donkey anti-rabbit Ig. The primary antibody was incubated with the beads for 1 hr on ice, washed twice, followed by an additional 1 hr with the labeled secondary. The beads were again washed and analyzed. FITC-annexin 5 (BD Biosciences) was used as described by the manufacturer. Samples included BSA-blocked beads and exosome-beads incubated in the presence and absence of Ca²⁺ (1 mM).

Brownlee Z., Lynn K.D., Thorpe P.E, & Schroit A.J. (2014). A novel “salting-out” procedure for the isolation of tumor-derived exosomes. Journal of immunological methods, 407, 120-126.

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Osteoclast Protein Lysate Analysis

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Cell protein lysates were harvested from osteoclasts grown in 12 well dishes (Corning) in modified RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM EDTA) supplemented with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Lysates were cleared by centrifugation at 12,000Xg at 4°C. Proteins were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore), subjected to western blotting by standard protocols, and visualized using ECL Prime (G.E. Health Systems). Blots were blocked and incubated at 4°C with primary antibodies diluted in TBS/0.1% Tween-20 plus 3% bovine serum albumin. Primary antibodies used were HDAC7-Abcam (Ab12174) 1:2000 dilution; ^α-TUBULIN- Santa Cruz (SC-5546) 1:1000 dilution; MYC- Santa Cruz (SC-40) 1:1000 dilution; MITF- Abcam (Ab12039) 1:1000 dilution; ACTIN- Santa Cruz (SC-1616) 1:2,000 dilution; FLAG—Sigma-Aldrich (F1804) 1:5,000 dilution. Horseradish-peroxidase conjugated secondary antibodies were diluted in TBS/0.1% Tween-20 plus 5% nonfat dry milk. Secondary antibodies used were from G.E. Health Systems: Amersham ECL anti-mouse (NA-931) and anti-rabbit (NA-934) at 1:8000, or Santa Cruz: anti-goat (SC-2020) at 1:12,000 dilution.

Stemig M., Astelford K., Emery A., Cho J.J., Allen B., Huang T.H., Gopalakrishnan R., Mansky K.C, & Jensen E.D. (2015). Deletion of Histone Deacetylase 7 in Osteoclasts Decreases Bone Mass in Mice by Interactions with MITF. PLoS ONE, 10(4), e0123843.

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Quantitative Immunoblotting of Autophagy Proteins

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Proteins (30 µg) extracted from the heart tissue samples were separated on 10% polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Hoffman-La Roche Ltd., Basel, Switzerland). The membranes were then incubated using antibodies against Rabbit LC3 (16–18 kDa; 1:1,000, L8918; Sigma-Aldrich Co.), Rabbit SQSTM1 (62 kDa; 1:1,000, SC-25575; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and beclin 1 (60 kDa; 1:1,000, #3738; Cell Signaling Technology, Inc., Danvers, MA, USA), after which the blots were visualized using enhanced chemiluminescence (RPN 2232; Amersham/GE Healthcare, Uppsala, Sweden). β-actin (43 kDa; 1:5,000, SC-5546; Santa Cruz Biotechnology Inc.) served as the loading control. The molecular band intensity was determined as a ratio relative to that of loading control.

Yuan X., Xiao Y.C., Zhang G.P., Hou N., Wu X.Q., Chen W.L., Luo J.D, & Zhang G.S. (2016). Chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice. Drug Design, Development and Therapy, 10, 2729-2737.

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Protein Signaling Pathway Analysis

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The following antibodies were used: EGFR (sc-03-G), Akt1 (sc-1618), C14ORF37 (UT2) (sc-139226), Rictor (sc-271081), Raptor (sc-81537) pAMPK (S485/491), AMPK (sc-25793), Alpha-Tubulin (sc-5546), were purchased from Santa Cruz Biotechnology. pEGFR (Y1173) from Invitrogen, Myc-tag (Cat#2278S), mTOR (Cat#2972), phospho p70S6K1 (T389) (Cat#9204), pPKC (T514) (Cat#9379), pAkt (S243) (Cat#4060), phospho MAPK (Cat#9101S), MAPK (Cat#4695), ULK1 (Cat#8054), pULK1-S757 (Cat#6888), and pULK1-(Cat#5869) were purchased from Cell Signaling. Actin (Cat#A2228) was from Sigma. PKC (Cat# ab71558) and phospho-mTOR(Cat#ab109268) were from Abcam. C225 (Cat #MABF120), was from EMD Millipore for EGFR immunoprecipitation studies. LC3 (Cat#NB100-2220) was from Novus biologicals. EGFR TKI AEE788 (Cat# S1486), Akt inhibitor, MK2206 (Cat#S1078), were obtained from Selleckchem. Plasmid-based transfections and siRNA-based transfections were performed using Lipofectamine 3000 (Invitrogen) and Lipofectamine RNAiMax (Invitrogen), respectively. Protein A/G beads (Santa Cruz Biotechnology) were used for immunoprecipitation.

Katreddy R.R., Bollu L.R., Su F., Xian N., Srivastava S., Thomas R., Dai Y., Wu B., Xu Y., Rea M.A., Briggs J.M., Zhang Q., Lu X., Huang G, & Weihua Z. (2018). Targeted reduction of the EGFR protein, but not inhibition of its kinase activity, induces mitophagy and death of cancer cells through activation of mTORC2 and Akt. Oncogenesis, 7(1), 5.

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Quantification of Syndecan-1 Protein Levels in Tissue Lysates

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Total protein concentration in tissue lysates was measured using bicinchoninic acid protein assays, and 10 μg of protein was separated by 10% SDS‐PAGE and transferred onto nitrocellulose membranes (Millipore Sigma, Billerica, MA). Membranes were probed with antibodies against syndecan‐1 (ab34164; Abcam, Cambridge, UK) or α‐tubulin (sc‐5546; Santa Cruz Biotechnology, Dallas, TX), and immunoreactive bands were visualised using enhanced chemiluminescence (GE Healthcare UK). Signal intensities were quantified (as arbitrary units) using ImageJ. Western blotting was performed on five independent samples from individual mice from each group.

Suzuki K., Okada H., Takemura G., Takada C., Tomita H., Yano H., Muraki I., Zaikokuji R., Kuroda A., Fukuda H., Nishio A., Takashima S., Suzuki A., Miyazaki N., Fukuta T., Yamada N., Watanabe T., Doi T., Yoshida T., Kumada K., Ushikoshi H., Yoshida S, & Ogura S. (2020). Recombinant thrombomodulin protects against LPS‐induced acute respiratory distress syndrome via preservation of pulmonary endothelial glycocalyx. British Journal of Pharmacology, 177(17), 4021-4033.

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Protein Expression Analysis Protocol

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Protein expression was analyzed as described (Wittmann, et al. 2007 (link)) using antibodies purchased from Sigma - A5441 (β-actin) and Santa Cruz Biotechnology (Santa Cruz, CA, USA) - sc-6259 (cytokeratin 18), sc-20680 (lamin A), sc-5546 (α-tubulin) and sc-8005 (ESR1).

Wardell S.E., Nelson E.R., Chao C.A., Alley H.M, & McDonnell D.P. (2015). Evaluation of the pharmacological activities of RAD1901, a selective estrogen receptor degrader. Endocrine-related cancer, 22(5), 713-724.

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Immunofluorescence Staining of CHOP in Frozen Liver

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Frozen liver sections (8-μm) were fixed in acetone (−20 °C for 20 min) and washed with tap water in a running type, then slides were incubated a blocking buffer (5% normal serum in 0.3% Triton™ X-100 in a 10 mM PBS) for 40 min. Then, mouse-anti-CHOP (CCAAT-enhancer-binding protein homologous protein) antibody (diluted in 1:100, #2895, Cell Signaling) with the antibody dilution buffer (1% BSA with 0.3% Triton™ X-100 in 10 mM PBS) was applied and incubated overnight. After incubation, the rabbit-α-tubulin antibody (diluted 1:1000, #sc-5546, Santa Cruz, Dallas, TX, USA) further incubated for overnight at RT. After washing the primary antibodies, secondary goat anti-mouse or rabbit IgG (H+L) secondary antibodies (Goat-anti-mouse Alexa Fluor^® 488 conjugate and Goat-anti-rabbit-Alexa Fluor^® 594 conjugate) was applied. The cell-based immunofluorescence analyses were conducted according to the manufacture’ protocol against CHOP. All histopathological features were examined under microscopy circumstance (200× or 400× magnification, IX71; Olympus, Tokyo, Japan).

Kim H.G., Wang J.H., Kim H.S., Lee J.S., Im H.J., Lee S.B., Lee D.S., Hur G.M, & Son C.G. (2021). Animal Evidence for Synergistic Induction of Hepatic Injury by Dietary Fat and Alcohol Consumption and Its Potential Mechanisms. Journal of Personalized Medicine, 11(4), 287.

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