Cells were lysed as previously described [20 (link)]. Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures [21 (link)]. Primary antibodies were Anti-GLI1 (L42B10, Cell Signalling Technology Inc., Boston, MA, USA), anti-PARP p85 Fragment (G7341, Promega, Madison, WI, USA) and anti-Cleaved NOTCH1 (D3B8, Cell Signalling Technology Inc., Boston, MA, USA). HRP-conjugated secondary antisera (Santa Cruz Biotechnology, Shanghai, China) were used, followed by enhanced chemiluminescence (ECL Amersham, Merk Life Science S.r.l., Milan, Italy).
Hrp conjugated secondary antisera
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
HRP-conjugated secondary antisera are enzyme-labeled antibodies used to detect primary antibodies in various immunoassays. These reagents contain horseradish peroxidase (HRP) conjugated to secondary antibodies that bind to the primary antibodies of interest.
Automatically generated - may contain errors
Lab products found in correlation
Anti gli1 l42b10, by Cell Signaling Technology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Anti mouse il 1β antibody, by R&D Systems (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Amersham ecl prime wb detection reagent, by GE Healthcare (1 mentions)
H5147, by Merck Group (1 mentions)
Image 6 quant las 500, by GE Healthcare (1 mentions)
Ac133, by Miltenyi Biotec (1 mentions)
Anti p21 c 19, by Santa Cruz Biotechnology (1 mentions)
Anti bax n 20, by Santa Cruz Biotechnology (1 mentions)
Anti gli1, by Novus Biologicals (1 mentions)
Anti bcl 2 n 19, by Santa Cruz Biotechnology (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Rabbit anti oct4, by Cell Signaling Technology (1 mentions)
Rabbit anti pcna, by Cell Signaling Technology (1 mentions)
Rabbit anti hsp60, by Cell Signaling Technology (1 mentions)
Mouse anti gapdh, by Abcam (1 mentions)
9 protocols using hrp conjugated secondary antisera
1
Western Blot Analysis of GLI1, PARP, and NOTCH1
2
Western Blot Analysis of IL-1β
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Supernatants, lysates, and pellet samples were separated by SDS‐PAGE using 10% or 16% polyacrylamide gels. After electrophoresis, the separated proteins were transferred to PVDF membranes (Pall Co., NY, USA). The membranes were probed overnight at 4°C with anti‐mouse IL‐1β antibody (R&D Systems, MN, USA) or anti‐actin antibody (Santa Cruz Biotechnology, CA, USA). The membranes were further probed with HRP‐conjugated secondary anti‐sera (Santa Cruz Biotechnology) and visualized by ECL solution (Millipore, MA, USA) and a cooled CCD camera system (ATTO Technology, Tokyo, Japan).
3
Immunoblotting Analysis of CD133 and HSP70
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Cells were lysed as previously described [65 (link)] and immunoblotted using standard procedures. Membranes were incubated overnight at + 4 °C with primary antibodies against CD133 (AC133, Miltenyi Biotec, Bergisch Gladbach, DE) and HSP70 (H5147, Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated secondary antisera (Santa Cruz Biotechnology) were used, followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK). Immunoreactive bands were visualized using Amersham ECL Prime WB detection reagent (GE Healthcare Europe, Milan, Italy). Images were acquired using an Image 6 quant LAS 500 (GE Healthcare Europe), and densitometric analysis was performed using ImageJ software.
4
Western Blot Analysis of Apoptosis Regulators
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Cells were lysed with T-PER® Tissue Protein Extraction Reagent (# 78510; Pierce Biotechnology, Rockford, IL) added with protease inhibitors. Lysates were separated on pre-cast gels (Bio-Rad Laboratories; Hercules, CA) and immunoblotted using standard procedures. Anti-p21 (C19) (# sc-397; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bax (N20) (# sc493, Santa Cruz Biotechnology), anti Bcl-2 (N19) (# sc-492, Santa Cruz Biotechnology), anti-Gli1 (# NB600-600, Novus Biologicals, Littleton, CO, USA) and HRP-conjugated secondary antisera (Santa Cruz Biotechnology) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK). Densitometry calculations for western blot were calculated using ImageJ software, verifying for non-saturation and subtracting background. Band intensities were sampled three times and normalized against HSP90 (# 4874; Cell Signaling Technology Inc., Danvers, MA).
5
Protein Expression Analysis in Cells
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Cells were lysed in Tris-HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM, and protease inhibitors. Lysates were separated on 10% or 12% acrylamide gel and immunoblotted using standard procedures. Rabbit anti-OCT4, #2750 (Cell Signaling Technology Inc., Danvers, MA), rabbit anti-Nanog, PA1-097 (ThermoFisher Scientific, Rockford, IL), mouse anti-β-3-Tubulin (TU-20), #4466 (Cell Signaling Technology Inc.), mouse anti-GFAP, MAB360 (Merck Millipore, Darmstadt), rabbit anti-HspA1A, sc-33575 (Santa Cruz Biotechnology, CA), rabbit anti-PCNA, #13110 (Cell Signaling Technology Inc.), rabbit anti-NPM, #3542 (Cell Signaling Technology Inc.), mouse anti-GAPDH, ab8245 (AbCam, Cambridge, UK), rabbit anti-Hsp60, #D307 (Cell Signaling Technology Inc.), and HRP-conjugated secondary antisera (Santa Cruz Biotechnology, CA) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK) and images were acquired using BioRad ChemiDoc MP Imaging System (BioRad, Hercules, CA). Densitometric analysis was performed using the BioRad associated Image Lab Software (BioRad, Hercules, CA). Values are expressed as fold over internal control, represented by GAPDH or Hsp60, in the case of NPM, whose expressions were not significantly modulated in the proteome profiles.
6
Protein Expression Analysis Protocol
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Cells were lysed in Tris–HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM and protease inhibitors. Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures. Primary antibodies were: Anti‐GLI1 (L42B10, Cell Signalling Technology Inc), anti-PARP p85 Fragment (G7341, Promega, Madison USA), anti‐PCNA (D3H8P, Cell Signalling Technology Inc), Anti-ABCA2 (NBP1-20863, Novus Biological), anti-ABCB1 (MDR1, D-11; sc-55510 Santa Cruz Biotechnology, Inc.), anti-ABCB4 (P3II-26, Abcam), anti-ABCB7 (ab151992, Abcam), anti ABCC2 (MDR2, ab3373 M2 III-6, Abcam) anti- ABCG1 (NB400-132, Novus Biological) and anti‐ABCG2 H-70 (sc‐2582; Santa Cruz Biotechnology, Inc.). HRP‐conjugated secondary antisera (Santa Cruz Biotechnology) were used, followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK). Western blots shown in figures are representative of at least three different experiments. Uncropped images for western blots are shown in supplementary Figs. 5 and 6 as indicated in figure legends.
7
Western Blot Analysis of Gli1 Protein
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Forty-eight hours after transfection, cells were harvested and lysed in a solution containing Tris-HCl pH 7.6, 50mM deoxycholic acid, sodium salt 1%, NaCl 150mM, NP40 1%, EDTA 5mM, NaF 100mM and protease inhibitors. Lysates were separated on SDS-PAGE and immunoblotted using standard procedures. Anti-Gli1 (Santa Cruz Biotechnology), anti-actin (Santa Cruz Biotechnology) and HRP-conjugated secondary antisera (Santa CruzBiotechnology) were used. Bands were visualized by EZ-ECL detection reagents.
8
Western Blot Analysis of Pluripotency and Cytoskeletal Markers
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Cells were lysed in Tris–HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM, and protease inhibitors. Lysates were separated on an 8% acrylamide gel and immunoblotted using standard procedures. Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk and incubated overnight at 4 °C with the following antibodies: rabbit anti-Foxm1 (Santa Cruz Biotechnology, sc-502), rabbit anti-Hsp70, (Santa Cruz Biotechnology, sc-33575), mouse anti- βIIItub (MAB 1637 Millipore), goat anti-actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-Nanog (PA1–41577, Thermo Fisher), anti-α-Tubulin Antibody (T9026 SIGMA). HRP-conjugated secondary antisera (Santa Cruz Biotechnology) were applied and binding visualized by enhanced chemiluminescence (ECL Amersham).
9
Western Blot Analysis of Cellular Proteins
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Cells were lysed in Tris-HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM and protease inhibitors. For phospho p65, 2 mM Na orthovanadate and 5 mM Na butyrate were also added. Lysates were separated on 8% or 10% acrylamide gel and immunoblotted using standard procedures. Rabbit anti-Hsp70, sc-33575 (Santa Cruz Biotechnology, CA), rabbit anti-PCNA, #13110 (Cell Signaling Technology Inc., Danvers, MA); mouse anti-b3-tubulin (TU-20), #4466 (Cell Signaling Technology Inc, Danvers, MA); mouse anti-GFAP, MAB360 (Merck Millipore, Darmstadt), rabbit anti-p65, #3034S (Cell Signaling Technology Inc, Danvers, MA); rabbit anti-phospho p65 (P-p65), #3033S (Cell Signaling Technology Inc, Danvers, MA); mouse anti-GAPDH, ab8245 (AbCam, Cambridge, UK) and HRP-conjugated secondary antisera (Santa Cruz Biotechnology, CA) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK) and images were acquired using the BioRad ChemiDoc MP Imaging System (BioRad, Hercules, CA). Densitometric analysis was performed using the BioRad associated Image Lab Software (BioRad, Hercules, CA). Values are expressed as fold over internal control, represented by GAPDH, that does not change significantly in the proteome profiles.
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