Protein lysates were prepared as previously described [11 ]. Briefly, 50 mg of the liver was homogenized in 1 mL of cell lysis buffer, 1X proteinase inhibitor, and 1X PMSF. Samples were sonicated on power 4, thrice for 5 s each, and then, spun at 13,200 RPMs for 15 min at 4 °C. Protein concentrations were determined using a BCA reagent (23,227; Pierce™). Protein lysates were resolved in 3–8% Tris-Acetate Novex gel (Invitrogen) for NCOR1, SMRT/NCOR2, and beta-actin or 10% Bis-Tris gel (Invitrogen) for hdac3 and hsp90, and then, transferred to a nitrocellulose membrane. Blots were probed for NCOR1 using rabbit polyclonal anti-NCOR1 antibody (PHQQ; generated against a C-terminal NCoR peptide as previously described; diluted at 1:250), rabbit polyclonal anti-SMRTe (06–891; Millipore Sigma; diluted at 1:500), or rabbit polyclonal anti-HDAC3 (ab7030; abcam; diluted at 1:2000) overnight at 4 °C with mouse monoclonal anti-beta-actin antibody (MA191399; Fisher Scientific; diluted at 1:10,000) for NCOR1 and SMRT/NCOR2 control or mouse monoclonal anti-hsp90a/b antibody (sc-13119, Santa Cruz Biotechnology; 1:5,000). Goat anti-rabbit Alexa Fluor Plus 680 (A32734; Invitrogen) and goat anti-mouse Alexa Fluor Plus 488 (A32723; Invitrogen) were used as secondary antibodies and incubated at room temperature for 90 min. Images were captured on ChemiDoc Touch MP (Bio-rad).
Sc 13119
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-13119 is a laboratory instrument used for protein analysis. It is designed to perform western blotting, a technique used to detect and quantify specific proteins in a sample. The core function of Sc-13119 is to facilitate the separation, transfer, and detection of proteins from a complex mixture.
Automatically generated - may contain errors
Lab products found in correlation
Sc 47778, by Santa Cruz Biotechnology (4 mentions)
Anti hsp90 αβ, by Santa Cruz Biotechnology (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ab75014, by Cell Signaling Technology (2 mentions)
P yap, by Cell Signaling Technology (2 mentions)
Af3629, by Abcam (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Hsp90, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab7030, by Abcam (1 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch mp, by Bio-Rad (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Sc 53540, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Sc 516102, by Santa Cruz Biotechnology (1 mentions)
1 step ultra tmb blotting reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gsk 3β, by Cell Signaling Technology (1 mentions)
Rpn2106, by GE Healthcare (1 mentions)
26 protocols using sc 13119
1
Protein Lysate Preparation and Western Blotting
2
Extracellular Vesicle Marker Identification
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Proteins extracted from EVs were subjected to Western blot analysis to identify EV markers. Briefly, SDS-PAGE electrophoresis was performed in 10% polyacrylamide gels and then transferred onto the PVDF membrane. Then, membranes were blocked and incubated with primary antibodies Anti-HSP90 αβ and Anti Alix (1:500, SC-13119 and SC-53540, 1:500, respectively; Santa Cruz Biotechnology, Inc. Dallas, TX, USA), and Anti Flotilin-1 (1:500, BD-610821, Franklin lakes, NJ, USA). Then, membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:5000; SC-516102; Santa Cruz Biotechnology, Inc. Dallas, TX, USA). Finally, protein bands were visualized with a 1-Step Ultra TMB-Blotting reagent (37574, Thermo Scientific, Waltham, MA, USA).
3
Immunoprecipitation and Western Blotting Protocols
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Mouse tissues and cells were harvested and dissolved in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton-X-100, 0.5% NP-40, pH 7.6) or NP-40 buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, pH 7.4) containing protease and phosphatase inhibitor cocktail (GenDEPOT, Huston). Immunoprecipitation was performed with anti-Flag (F1804, Sigma) antibody overnight at 4 °C, followed by addition to protein G-Sepharose beads (Upstate Biotechnology) for two hours at 4 °C. Liver nuclear extracts were prepared as described previously 27 (link). Protein lysates were performed to Western blotting with the following primary antibodies [rat anti-HA (3F10, Roche), rabbit anti-GLUT2 (ab54460, Abcam), rabbit anti-β-actin (AbC-2004, Abclon), mouse anti-Flag (F1804, Sigma), mouse anti-HSP90α/β (sc-13119, Santacruz), rabbit anti-CREBH (EWS101, Kerafast), mouse anti-Lamin B1 (sc-377001, Santacruz), goat anti-PTP4A1(EB06456, Everest), and rabbit anti-GSK3 β (#12456, Cell Signaling Technology)]. The membranes were incubated with primary antibodies followed by the horseradish peroxidase-conjugated secondary antibodies (rat: 31470, rabbit: 31464, Thermo Fischer Scientific; mouse: AbC-5001, AbClon) for one hour at room temperature. Immuno-reactive bands were visualized using a chemiluminescent substrate (RPN2106, GE).
4
Antibody Sourcing for Cellular Analyses
5
Western Blot Analysis of Cellular Proteins
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Cells were collected and homogenized in radioimmunoprecipitation assay buffer. Total cell protein extracts were quantified and assessed by Western blot as described elsewhere (15 (link)). The following antibodies diluted in blocking solution [phosphate-buffered saline (PBS) and 0.1% Tween 20 containing 5% milk] were used: anti-HSP90 (1:5000; sc13119, Santa Cruz Biotechnology), anti-Flag (1:3000; F7425, Sigma-Aldrich), anti-TMBIM1 (1:1000; MBS1499661, MyBioSource), anti-BAX (1:1000; 06-499, EMD Millipore), anti-MYC (1:3000; ab9106, Abcam), and anti-actin (1:10,000; sc-8432, Santa Cruz Biotechnology). Bound antibodies were detected using peroxidase-coupled secondary antibodies and the enhanced chemiluminescence system.
6
Western Blot Analysis of Protein Samples
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Samples were prepared with reducing Laemmli buffer, denatured by heating at 95 °C for 10 min and subjected to SDS-PAGE. By semi-dry blotting, proteins were transferred onto a nitrocellulose membrane (#10600002, GE Healthcare Life Sciences, Uppsala, Sweden) that was then blocked with 5% nonfat dry milk in PBS-T (PBS containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were diluted in PBS-T as well and incubated accordingly. Antibodies used were directed against His6 (1:1.000, #MA1-21315, Invitrogen, Carlsbad, CA, USA), Hsp90 (1:1.000, #sc-13119, SantaCruz Biotechnology, Dallas, TX, USA) and GAPDH (1:1.000, #sc-365062, SantaCruz Biotechnology, Dallas, TX, USA), all incubated 1 h at room temperature. Anti-NDPK-A (1:200, #sc514515, SantaCruz Biotechnology, Dallas, TX, USA) was applied over night at 4 °C. For detection an anti-mouse IgG kappa binding protein (m-IgGκ BP) HRP-conjugate (1:2.500, #sc-516102, SantaCruz Biotechnology, Dallas, TX, USA) was used, diluted in PBS-T and incubated for 1 h at room temperature. For visualization, the ECL system (#WBKLS0500, EMD Millipore Corporation, Darmstadt, Germany) was used.
7
HNF6 Knockdown in Hepa-1c1c7 Cells
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24 h after seeding cells, HNF6-shRNA plasmid (HNF-6 sc-37937-SH, Santa Cruz Biotechnology, USA) was used for knocking-down of HNF6, according to the manufacturer's instructions. Scrambled shRNA plasmid (sc-108060, Santa Cruz Biotechnology) was used as a negative control. Knockdown efficiency was assessed 48 h post-transfection by western blotting as well as by real time PCR. Protein of transfected Hepa-1c1c7 cells was extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate containing protease and phosphatase inhibitors). Protein samples were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose (Amersham Protran Supported 0.45 NC, GE Healthcare). After blocking with 0.5% dry milk in PBS-Tween 0.1%, the membranes were probed with anti-HNF6 (1:500, Santa Cruz Biotechnology, sc-376167), and HSP90 (1:1000, Santa Cruz Biotechnology, sc-13119) antibodies overnight at 4°C. Anti-rabbit and -mouse HRP conjugated antibody was used as a secondary antibody. Detection of the immune complexes was performed using WesternBright Quantum system (Advansta, K-12042, USA) and quantification was done with the Quantity One analysis software (Bio-Rad).
8
Extracellular Vesicle Protein Profiling
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Equivalent amounts of total protein from extracellular vesicles and cells were separated using SDS–PAGE and transferred to a polyvinylidene fluoride membrane (88520, Thermo Fisher Scientific, Waltham, MA, USA). The membrane was blocked with 5% non-fat milk in 1X TBS-T (0.05%) for 2 h at room temperature and subsequently incubated with primary antibodies anti-HSP90 α/β (1:3000, sc-13119, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HSP70 (1:1000, sc-24, Santa Cruz Biotechnology), anti-CD9 (1:250, sc-13118, Santa Cruz Biotechnology), anti-Calnexin (1:400, sc-23954, Santa Cruz Biotechnology), anti-vitronectin (1:500, sc-74484, Santa Cruz Biotechnology), and anti-β-catenin (1:500, ab2365, Abcam, Cambridge, UK) overnight at 4 °C. The membranes were washed with TBS-T and incubated with a secondary antibody (1:5000, 115-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA, or 1:3000, 65-6120, Invitrogen, Waltham, MA, USA) for 2 h at room temperature. The membranes were washed with TBS-T three times and scanned on a C-DiGit Blot scanner (LI-COR Biosciences, Lincoln, NE, USA) using an Immobilon Crescendo Western HRP Substrate (WBLUR0100, Millipore, Burlington, MA, USA). Image Studio Digits v.5.2 software (LI-COR Biosciences, Nebraska, USA) was used for image acquisition.
9
Monitoring CDK4 Regulation Under ER Stress
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HEK293 cells were obtained from the American Type Culture Collection (ATCC) and cultured under standard conditions recommended by the ATCC. HEK293 cells were cultured on 60-mm dishes to 40 to 50% confluence, treated with tunicamycin for 72 hours at the indicated concentrations to induce ER stress, and harvested for immunoblotting with anti-CDK4 antibody.
For immunoprecipitation with anti-CDK4, HEK293 Ctrl or shCHIP cells were incubated with tunicamycin (1 μg/ml) for 24 hours and then treated with 10 μM MG132 for 1.5 hours. The cells were lysed by sonication in RIPA lysis buffer as described previously (13 (link)). Four milligrams of protein lysates was incubated with 20 μl of anti-CDK4 agarose (#sc-260, Santa Cruz Biotechnology) overnight at 4°C. The beads were washed with RIPA buffer three times, and 60 μl of 1× SDS-PAGE loading buffer was added. The sample was boiled for 5 min. Half of the immunoprecipitates or 50 μg of the total protein lysate was loaded for SDS-PAGE and assayed by Western blotting. The membranes were probed with anti-HSP70 (SC-24, Santa Cruz Biotechnology), anti-HSP90 (SC-13119, Santa Cruz Biotechnology), anti-CDC37 (SC-13129, Santa Cruz Biotechnology), anti-CHIP (SC-133066, Santa Cruz Biotechnology), and anti–α-tubulin antibody (T6199, Sigma-Aldrich).
For immunoprecipitation with anti-CDK4, HEK293 Ctrl or shCHIP cells were incubated with tunicamycin (1 μg/ml) for 24 hours and then treated with 10 μM MG132 for 1.5 hours. The cells were lysed by sonication in RIPA lysis buffer as described previously (13 (link)). Four milligrams of protein lysates was incubated with 20 μl of anti-CDK4 agarose (#sc-260, Santa Cruz Biotechnology) overnight at 4°C. The beads were washed with RIPA buffer three times, and 60 μl of 1× SDS-PAGE loading buffer was added. The sample was boiled for 5 min. Half of the immunoprecipitates or 50 μg of the total protein lysate was loaded for SDS-PAGE and assayed by Western blotting. The membranes were probed with anti-HSP70 (SC-24, Santa Cruz Biotechnology), anti-HSP90 (SC-13119, Santa Cruz Biotechnology), anti-CDC37 (SC-13129, Santa Cruz Biotechnology), anti-CHIP (SC-133066, Santa Cruz Biotechnology), and anti–α-tubulin antibody (T6199, Sigma-Aldrich).
10
Immunoblotting and Microscopy Protocols
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Antibodies used for immunoblotting: rabbit anti-ErbB2 (29D8, Cell Signaling), mouse anti-ErbB2 (Ab-17, Thermo Fisher Scientific), rabbit anti-ErbB3 (Ab-1328, Sigma Aldrich, MO), rabbit anti-Ebp50 (SLC9A3R1, Sigma Aldrich), rabbit anti-Ebp50 (A310, Cell Signaling), mouse anti-ezrin (E8897, Sigma Aldrich), rabbit anti-radixin (HPA000763, Sigma Aldrich), rabbit anti-pERM (3149, Cell Signaling), mouse anti-GAPDH (Ab-9484, Abcam), mouse anti-pErk1/2 (9106, Cell Signaling), rabbit anti-pAkt (4058, Cell Signaling), mouse anti-Ubiquitin (P4G7, Covance). Antibodies used for microscopical analysis: rabbit anti-ErbB2 (29D8, Cell Signaling), mouse anti-ErbB2 (9G6, Santa Cruz), rabbit anti-pERM (3149, Cell Signaling), rabbit anti-ezrin (3145, Cell Signaling), mouse anti-ezrin (E8897, Sigma Aldrich), rabbit anti-radixin (HPA000763, Sigma Aldrich), mouse anti-radixin (1E12, Novus Biologicals), rabbit anti-Ebp50 (SLC9A3R1, Sigma Aldrich), mouse anti-Hsp90 (sc-13119, Santa Cruz), mouse anti-c-Cbl (Upstate). HEPES, bovine serum albumin, geldanamycin, lactacystin, chloroquine and n-octylglucopyranoside were purchased from Sigma-Aldrich. The small molecule inhibitor NSC668394 was purchased from Calbiochem, and the dynamin-inhibitor Dyngo4a was purchased from Fisher Scientific. The plasmids encoding GFP-ErbB2 and CFP-ErbB3 were kind gifts from Dr. B. van Deurs (University of Copenhagen, Denmark).
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