Whole-cell lysates were generated by the addition of 2× Laemmli buffer containing 10% β-mercaptoethanol to the cell pellets. Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blotted with the following antibodies: anti-caspase-8 (1C12; Cell Signaling), anti-caspase-3 (8G10; Cell Signaling), anti-PCNA (D3H8P; Cell Signaling), and anti-β-actin (AC-15; Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were used (BioLegend). Membranes were developed using enhanced chemiluminescence (ECL) (Thermo Scientific) and detected using a Nikon camera as previously described (82 (link)). Quantification analysis of blots was performed using ImageJ, and the β-actin signal was used as a control for normalization. Values are expressed as a percentage of the normalized signal from the mock-infected group.
Anti caspase 3 8g10
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-caspase-3 (8G10) is a mouse monoclonal antibody that recognizes human caspase-3. Caspase-3 is a cysteine protease that plays a central role in the execution phase of cell apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Anti caspase 8 1c12, by Cell Signaling Technology (1 mentions)
Anti pcna d3h8p, by Cell Signaling Technology (1 mentions)
Anti β actin ac 15, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibodies, by BioLegend (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti hes1, by Abcam (1 mentions)
Anti ph3, by Cell Signaling Technology (1 mentions)
Anti c caspase3, by Cell Signaling Technology (1 mentions)
Anti parp 46d11, by Cell Signaling Technology (1 mentions)
Anti ki67 b56, by BD (1 mentions)
Anti 8 ohdg, by Abcam (1 mentions)
Anti lc3a b d3u4c, by Cell Signaling Technology (1 mentions)
Anti dclk1, by Abcam (1 mentions)
Anti claudin 1, by Cell Signaling Technology (1 mentions)
Anti chga, by Abcam (1 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti brg1 epncir111a, by Abcam (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti caspase 3 8g10
1
Western Blot Analysis of Apoptosis Markers
2
Comprehensive Antibody Characterization Protocol
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The primary antibodies used in this study were as follows: anti-BRG1 (EPNCIR111A) Abcam Cat# ab110641 (1:10,000); anti-Caspase3 (8G10), Cell Signaling Technology, Cat# 9665 (1:1000); anti-C-Caspase 3, Cell Signaling Technology, Cat# 9661 (1:1000); anti-PARP (46D11), Cell Signaling Technology, Cat# 9532 (1:1000); anti-Ki67 (B56), BD Biosciences, Cat# 550609 (1:500); anti-EpCAM (G8.8), BD Biosciences, Cat# 552370 (1:500); anti-ZO1, Invitrogen, Cat# 40-2200 (1:500); anti-8-OHd G, Abcam, Cat# ab48508 (1:500); anti-LC3A/B (D3U4C), Cell Signaling Technology, Cat# 12741 (1:1000); anti-ATG16L1 (D6A5), Cell Signaling Technology, Cat# 8089 (1:1000); anti-PCNA, Santa Cruz Biotechnology, Cat# SC-7909 (1:1000); anti-P-H3, Cell Signaling Technology, Cat# 9701 (1:1000); anti-E-Cadherin, Cell Signaling Technology, Cat# 3195T (1:1000); anti-DCLK1, Abcam, Cat# ab31704 (1:500); anti-Claudin1, Cell Signaling Technology, Cat# 4933T (1:1000); anti-ChgA, Abcam, Cat# ab715 (1:1000); anti-Hes1, Abcam, Cat# ab71559 (1:500). Raw data of immunoblotting can be found in the source data file.
3
Evaluation of Epithelial-Mesenchymal Transition Markers
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The primary antibodies used anti-E-cadherin (Clone 36; BD Transduction Laboratories, San Jose, CA, USA), anti-ZEB1 (H-102; Santa Cruz), anti-actin (C-2; Santa Cruz), anti-FLASH (M-300; Santa Cruz), anti-caspase-3 (8G10; Cell Signaling Technology Inc, Danvers, MA, USA), anti-cleaved PARP-1 (D214; Cell Signaling), anti-lamin A/C (4C11; Cell Signaling), anti-ubiquitin (P4D1; Cell Signaling), anti-phospho-SMAD2 (D27F4; Cell Signaling), anti-SMAD2/3 (D7G7; Cell Signaling), anti-SNAIL (C15D3; Cell Signaling) and anti-SLUG (C19G7; Cell Signaling) were obtained commercially. Secondary antibodies horseradish peroxidase-conjugated anti-mouse and anti-rabbit (1:5000) were from Jackson Laboratories (West Grove, PA, USA). Gemcitabine was purchased from Tocris (Bristol, UK). MG132 and TGFβ were purchased from Cell Signaling.
4
Inhibition of p300 Enhances Gemcitabine-Induced Apoptosis
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Culture media (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were obtained from Gibco (Grand Island, NY). Anti-p300 (N-15; sc-584) and anti-SP1 (PEP2; sc-59) antibodies were obtained from Santa Cruz Biochemistry (Santa Cruz, CA). Anti-Caspase3 (8G10; #9662), anti-cleaved Caspase3 (5A1E; #9664), anti-Caspase8 (8G10; #9662), anti-Caspase9 (8G10; #9662), anti-cleaved PARP (D64E10; #5625), anti-γ-H2AX (20E3; #9718), anti-Acetyl Histone H3 (Lys9) (C5B11; #9649), anti-Acetyl Histone H3 (Lys27) (D5E4; #8173), and anti-Histone H3 (D1H2; #4499) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-β-actin antibody (AC-15; A5441) was obtained from Sigma-Aldrich (St. Louis, MO). Gemcitabine hydrochloride (G6423) and the p300 inhibitor C646 (SML0002) were obtained from Sigma-Aldrich.
5
Catechins and Apoptosis Induction
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Catechins (Pharmanex, USA, each capsule contains EGCG 95 mg, ECG 37 mg, EGC 15 mg), EGCG (Sigma-ALDRICH, E4143, C22H18O11, MW: 458.37), ECG (Shanghai Winherb Medical Technology Co., Ltd, C22H18O10, MW: 442.37) and EGC (Shanghai Winherb Medical Technology Co., Ltd, C15H14O7, MW: 306.27) were prepared at the concentration of 10 mM with RPMI 1640 medium (GIBCO-BRL, Grand Island, NY, USA). Arsenic trioxide (As2O3, Sigma-ALDRICH, A1010, MW: 197.84) was dissolved in RPMI 1640 medium as 5 mM solution. All-trans-retinoic acid (ATRA, Sigma-ALDRICH) was dissolved in ethanol as 100 μM solution. Pan-caspase inhibitor ZVAD-FMK (627610) was from Merck & Co. Inc. Bortezomib was from Millennium Pharmaceuticals (Cambridge, MA, USA). The primary antibodies against β-actin (13E5, #4970), anti-caspase-3 (8G10, #9665), anti-caspase-8 (D35G2, #4790), anti-caspase-9 Antibody (#9502), anti-PARP Antibody (#9542), anti-Bcl-2 (D55G8, #4223), anti-Bax (D2E11, #5023), anti-Bcl-xL (54H6, #2764) were from Cell Signaling (Beverly, MA, USA). Mouse monoclonal anti-PML Protein (ab57276) was from Abcam (Hongkong). The secondary antibody ImmunoPure Goat Anti-Rabbit IgG (#31460) was from Thermo Scientific (USA). Goat anti-mouse IgG (PB001) was from Shanghai Immune Biotech Ltd (ImB, CHINA). Chemiluminescence phototope-horseradish peroxidase kit (WBKLS0100) was from Millipore (Germany).
6
Analysis of UBE2L6 regulation and function
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The authenticity of Myc-VCP, Myc-UBE2L6 and HA-ISG15 was confirmed by DNA sequencing. The siRNA targeting UBE2L6 (sc-41685) and siRNA control (sc-37007) were purchased from Santa Cruz Biotechnology, Inc., CA, USA. Western blot and immune-fluorescence were done according to standard protocols. Antibodies and fluorescent dye used in this study are anti-UBE2L6 (ab71800, Abcam), anti-Tubuline (T8203, Sigma), anti-Caspase 3 (8G10, Cell Signaling Technology, Beverly, MA, USA), anti-GAPDH (6C5, Santa Cruz), anti-Ki-67 (610969, BD Biosciences, San Diego, CA), anti-VCP (ab109240, Abcam), anti-ATGL (F7, Santa Cruz), anti HA (ab18181, Abcam). Secondary antibodies are anti-Rabbit/mouse IgG-HRP conjugate (Bio-Rad), Alexa fluor 594 goat anti-rabbit IgG (H+L), Alexa fluor 488 goat anti-mouse IgG (H+L) (Invitrogen). DAPI was from Beyotime (Hangzhou, China). IFN-αwas purchased from Life technologies (PHC4014, Grand Island, NY, USA).
The blots were visualized using enhanced chemiluminescent detection (Pierce, Rockford) with image detection on XDBF-1 film (Eastman Kodak, Rochester, NY, USA). Immuno-fluorescence images were taken on a Nikon A1 confocal microscope (Nikon, Tokyo, Japan).
The blots were visualized using enhanced chemiluminescent detection (Pierce, Rockford) with image detection on XDBF-1 film (Eastman Kodak, Rochester, NY, USA). Immuno-fluorescence images were taken on a Nikon A1 confocal microscope (Nikon, Tokyo, Japan).
7
Antibodies for Protein Analysis
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Antibodies used in this study were anti-α-tubulin (DM1A, Sigma-Aldrich), anti-Caspase-7 (C7, Cell Signaling), anti-Caspase-3 (8G10, Cell Signaling), anti-GFP (clone B-2, Santa Cruz and ab1218, Abcam), hnRNP I (sc-133667, Santa Cruz), anti-PRPF6 (sc-48786, Santa Cruz), anti-PRPF8 (ab87433, Abcam), anti-TAP (PAP) (Sigma-Aldrich), anti-hSnu66 (A301–423A, Bethyl), anti-SC35 (for immunofluorescence: ab11826, Abcam; for immunoblotting: #556363, BD Biosciences), anti-Son (HPA023535, Sigma-Aldrich), anti-2,2,7-trimethylguanosine (K121, Calbiochem), anti-U1A (ab55751, Abcam), anti-U2AF65 (ab37483, Abcam), anti-Sm antigen Y12 (ab3138, Abcam). For immunofluorescence Alexa Fluor 488- and Alexa Fluor 555-labeled secondary goat anti-mouse and donkey anti-rabbit/anti-mouse antibodies from Invitrogen were used. Hub1-specific antibodies against recombinant S. cerevisiae Hub1 and human Hub1, respectively, were affinity-purified from serum of immunized rabbits.
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