The following antibodies were used for Western blot (WB) anlysis: Anti-MYC clone 9E10 (1:5000, Millipore-Sigma, M4439), Anti-MYC clone Y69 (1:1000, Abcam, Ab32072), Anti-Exportin-1 clone D6V7N (1:1000, Cell Signaling Technology, 46249), Anti-GAPDH clone 14C10 (1:5000, Cell Signaling Technology, 2118), Anti-alpha Tubulin clone DM1A (1:2000, Millipore-Sigma, T9026), Anti-beta Actin (1:1000, Cell Signaling Technology, 4967), Anti-rabbit IgG IRDye800CW (1:10000, Licor, 926-32211), Anti-mouse IgG IRDye680RD (1:10000, Licor, 926-68070), Anti-rabbit IgG-AP (1:5000, Invitrogen, G-21079), Anti-mouse IgG/IgM-AP (1:5000, Invitrogen, 31330). The following antibodies were used for immunohistochemistry (IHC): Anti-MYC clone EP121 (1:150, Millipore-Sigma, 395 R), Anti-cleaved Caspase-3 (Asp175) (1:100, Cell Signaling Technology, 9661), Biotinylated anti-rabbit IgG (1:500, Vector Laboratories, BA-1000-1.5). For immunofluorescence (IF) stainings, we used the following antibodies: Anti-phospho Histone-3 (Ser10) (1:200, Cell Signaling Technology, 9701), Anti-cleaved Caspase-3 (Asp175) (1:100, Cell Signaling Technology, 9661), Anti-rabbit IgG AlexaFluor568 (1:400, Invitrogen, A-11011). Immunofluorescence or bright field images were taken with 20x objectives on a Leica DMI6000 B microscope and quantified using MetaMorph (v7.8) image analysis software.
Anti cleaved caspase 3 asp175
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-cleaved caspase-3 (Asp175) is a laboratory reagent that specifically detects the cleaved form of caspase-3, a protein involved in the apoptosis (programmed cell death) pathway. This antibody recognizes the p17 fragment of activated caspase-3, which is generated upon cleavage at aspartic acid 175. It can be used to monitor apoptosis in experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (8 mentions)
Anti caspase 3, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (4 mentions)
Anti caspase 9, by Cell Signaling Technology (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Anti ki67, by Cell Signaling Technology (3 mentions)
Anti actin, by Merck Group (3 mentions)
Anti phospho histone h3 ser10, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti gfp, by Thermo Fisher Scientific (3 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (3 mentions)
Anti parp, by Cell Signaling Technology (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti ki67, by Abcam (3 mentions)
Anti cleaved caspase 9 asp315, by Cell Signaling Technology (3 mentions)
Anti erk1 2, by Cell Signaling Technology (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
84 protocols using anti cleaved caspase 3 asp175
1
Western Blot and Immunostaining Antibody Panel
2
Whole-mount Immunofluorescence Imaging of Xenografts
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Whole-mount immunofluorescence was performed starting hydration through methanol series (75% > 50% > 25%). Next, xenografts were permeabilized with 0.1% (w/v) Triton in PBS and blocked with a mixture of PBS 1X, BSA, DMSO, Triton 1% (w/v), and goat serum, for 1 h at room temperature. The xenografts were then incubated with primary antibody Anti-Cleaved caspase-3 (Asp175) (rabbit, Cell Signaling, 1:100, #9661) or Mpx (rabbit, GeneTex, 1:50, #gtx128379) overnight and followed by incubation of the secondary antibody goat anti-rabbit IgG (H+L) 650 (Dylight, 1:400, #84546) and 50 μg/ml DAPI (for nuclear counterstaining), again overnight.
Wash and fixation steps were performed, and xenografts mounted between two coverslips, allowing double side acquisition using Mowiol mounting media (Sigma).
Wash and fixation steps were performed, and xenografts mounted between two coverslips, allowing double side acquisition using Mowiol mounting media (Sigma).
3
Immunostaining of Apoptosis-Related Proteins
4
Analyzing Cell Death Rates in NRCM
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To analyze cell death rates, NRCM were transduced with Ad.Control or Ad.TIP30 adenovirus, stimulated with PE for 48 h, washed with 1× PBS, and incubated with 7‐AAD (Annexin V: PE Apoptosis Detection Kit I, BD Biosciences) 1:400 in 1× PBS for 15 min. After fixation with 100% ethanol, cells were mounted with VECTASHIELD Mounting Medium (Vector Laboratories) with DAPI. Tissue staining of cleaved caspase‐3 (CC3), the activated form of caspase‐3, was done with rabbit polyclonal anti‐Cleaved Caspase‐3 (Asp175, #9661, Cell Signaling, 1:100) using standard procedures.
5
Co-immunoprecipitation Assay for Protein-Protein Interactions
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Cells were lysed in 20 mM Hepes buffer, 10 mM KCl, 1 mM EDTA, 0.2% NP-40, 10% Glycerol44 (link), 59 (link) and co-immunoprecipitation studies were performed as previously described.44 (link) Details can be found in Supplementary Information . The following antibodies were used: mouse monoclonal anti-β-Actin (AC-15, Sigma-Aldrich, St Louis, MO, USA), mouse anti-HSP90 (Heat Shock Protein 90), rabbit polyclonal anti-KLF4 (sc-20691), rabbit polyclonal anti-p21 (C-19; sc-397), mouse monoclonal anti-p53 (DO-1; sc-126), mouse anti-Myc (9E10), mouse monoclonal anti-Caspase-3 (E-8; sc-7272), goat anti-Fibrillarin (D-14; sc-11336), goat anti-GAPDH (V-18; sc-20357), and mouse anti-BRAF wt (sc-5284) (Santa Cruz Biotechnology), rabbit polyclonal anti-BAX, rabbit polyclonal anti-BCL2, rabbit polyclonal anti-E2F1, rabbit polyclonal anti-poly ADP-ribose polymerase, rabbit polyclonal anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), rabbit polyclonal anti-Cleaved Caspase 3 (Asp175) (Cell Signaling Technologies, Danvers, MA, USA). Chemiluminescent detection was used.
6
Histological Analysis of Tumor Samples
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA buffer (R0010, Solarbio, Beijing, China). Protein extracts were transferred to PVDF membranes, and then incubated using appropriate antibodies. An ECL imaging system (Tanon-5200, Shanghai, China) was used to detect ECL signals. The primary antibodies used were: anti-LTB4R (ERP7113, 1:1000, Abcam, Carlsbard, CA, USA); anti-GAPDH (A19056, 1:1000, ABclonal, Hubei, China), anti-CDK2 (A0094, 1:1000, ABclonal), anti-CDK4 (A11136, 1:1000, ABclonal), anti-cyclin D1 (A19038, 1:1000, ABclonal), anti-Bcl-2 (A19693, 1:1000, ABclonal), anti-Bax (A19684, 1:1000, ABclonal), anti-caspase3 (9662, 1:1000, Cell Signaling Technology), anti-cleaved-caspase3 (Asp175, 1:1000, Cell Signaling Technology), anti-vimentin (A19607, 1:1000, ABclonal), anti-NCA (A19083, 1:1000, ABclonal), anti-ECA (A18135, 1:1000, ABclonal), anti-AKT (A17909, 1:1000, ABclonal), anti-P-AKT (AP0637, 1:1000, ABclonal), anti-mTOR (A11355, 1:1000, ABclonal), and anti-P-mTOR (AP0115, 1:1000, ABclonal). The second antibody was Anti-Rabbit-IgG(H+L)-HRP (AS030, 1:10,000, ABclonal).
8
Western Blot Protein Analysis Procedure
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Western blot analysis was carried out as described (Corno et al., 2017 (link)). Briefly, samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk, and then incubated overnight at 4°C with the following antibodies: anti-phospho-Akt (Ser473), anti-Akt (BD Science, Franklin Lakes, NJ, United States), anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), anti-ERK1/2, anti-AR (Millipore, Burlington, MA, United States); anti-Hsp90 (ac-Lys294) (Novus, Centennial, Colorado, United States), anti-Hsp90 (Santa Cruz Biotechnology, Dallas, TX, United States), anti-acetylated alfa-tubulin (Sigma-Aldrich, Milan, Italy), anti-Bax and anti-FLIPL (Sigma-Aldrich, Milan, Italy), anti-p53 (Dako, Santa Clara, CA, United States), anti-cleaved caspase-3 (Asp175) and anti-cleaved caspase-7 (Asp198) (Cell Signaling, Danvers, MA, United States). Anti-vinculin (Sigma-Aldrich, Milan, Italy), anti-β-tubulin (Abcam, Cambridge, United Kingdom) or anti-actin (Sigma) antibodies were used as control for loading. Antibody binding to blots was detected by chemo-luminescence (Amersham Biosciences, Cologno Monzese, Italy). Three independent experiments were performed.
9
Antibody Panel for Protein Expression Analysis
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The mouse monoclonal antibodies, namely anti-PR (sc-166169), anti-ERβ (sc-53494), anti-c-Jun (sc-74543), anti-p-c-Jun (sc-822), anti-JNK (sc-7345), anti-p-JNK (sc-6254), anti-C/EBPβ (sc-7962), and rat monoclonal antibody anti-ERα (sc-53493) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The rabbit polyclonal antibodies, including anti-phospho-cdc2 (Tyr15) (#9111), anti-cdc2 (#9112), anti-cleaved caspase 3 (Asp175) (#9661), anti-caspase 3 (#9662); rabbit monoclonal antibodies, including anti-phospho-cdc25C (Ser216) (#4901), anti-cdc25C (#4688), anti-phospho-histone H3 (Ser10) (#3377), anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (#2118), anti-PR A/B (#3153), anti-FOXO1 (#2880), and anti-IGFBP1 (#31025) antibodies; and the anti-rabbit (#7074) and anti-mouse (#7076) IgG, horse radish peroxidase (HRP)-linked antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The mouse monoclonal anti-Ki-67 (M7240) was purchased from Dako (Agilent; Santa Clara, CA, USA). All antibodies were used at the concentrations recommended by the manufacturers.
10
Cleaved Caspase Detection by Western Blot
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Protein extracts (50 µg) were fractionated by 10% SDS-PAGE and then transferred to PVDF membranes. The blots were washed with wash buffer (PBS1x, 0.05% Tween20), blocked for 1 h with 0.1% BSA, and then incubated with primary antibodies anti-cleaved Caspase-1 (Asp296) from Cell Signaling and anti-cleaved Caspase 3 (Asp175) (cat nº 96645) from Cell Signaling. The protein levels were expressed as the ratio between the target protein and ß-actin or tubulin detected in the same membrane.
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