Cell lysates were prepared by resuspending cell pellets in 1× Laemmli sample buffer containing 5% β-mercaptoethanol. Protein from lung tissues were extracted using IPH lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40) containing protease inhibitor cocktail (Roche). The protein lysates were separated by SDS–PAGE and then electrotransferred to nitrocellulose membranes (Millipore Corp., Bradford, MA, USA). Detection of specific proteins was carried out with enhanced chemiluminescence reagents following the manufacturer’s instructions (P90720, Millipore Corporation, MA, USA). The primary antibodies used for western blotting were as follows: anti-Caspase-9 (#9508), anti-Caspase-8 (#9746), anti-Caspase-7 (#8438), anti-Caspase-3 (#9664), anti-Bim (#2933), anti-Bak (#3814), anti-Bcl-xL (#2762), anti-p-Jak2 (#3771), anti-Jak2 (#3230), and anti-p-STAT3Y705 (#9145), which were purchased from Cell Signaling Technology. Anti-CDK2 (sc-6248), anti-p53 (sc-126), anti-STAT3 (sc-8019), anti-PARP-1 (sc-74470), anti-Cyclin D1 (sc-8396), and anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. An anti-p21 (ab7960), anti-p16 (ab108349), anti-IL-6 (ab6672), and anti-TNF-α (ab6671) antibodies were purchased from Abcam. Densitometry analyses were performed using ImageJ software (National Institutes of Health, NIH).
Anti bim
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Bim is a primary antibody product manufactured by Cell Signaling Technology. It is designed to detect the Bim protein, which is a member of the Bcl-2 family and plays a role in apoptosis, or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax, by Cell Signaling Technology (21 mentions)
Anti bcl xl, by Cell Signaling Technology (19 mentions)
Anti akt, by Cell Signaling Technology (16 mentions)
Anti cleaved parp, by Cell Signaling Technology (15 mentions)
Anti bcl 2, by Cell Signaling Technology (15 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (15 mentions)
Anti mcl 1, by Cell Signaling Technology (14 mentions)
Anti puma, by Cell Signaling Technology (13 mentions)
Anti caspase 3, by Cell Signaling Technology (12 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (12 mentions)
Anti bak, by Cell Signaling Technology (11 mentions)
Anti β actin, by Merck Group (10 mentions)
Anti erk, by Cell Signaling Technology (9 mentions)
Anti phospho erk, by Cell Signaling Technology (8 mentions)
Anti parp, by Cell Signaling Technology (8 mentions)
Anti xiap, by Cell Signaling Technology (7 mentions)
Anti bax, by Santa Cruz Biotechnology (7 mentions)
Anti chop, by Cell Signaling Technology (7 mentions)
Anti caspase 9, by Cell Signaling Technology (6 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (6 mentions)
89 protocols using anti bim
1
Western Blotting Analysis of Apoptosis Markers
2
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed in lysis buffer (1% NP‐40, 150 mM NaCl, 20 mM Tris‐HCl, pH 7.5, 0.5 mM EGTA, and 0.1 mM DTT) containing cOmplete mini protease inhibitor cocktail tablets (Merck) and phosphatase inhibitor cocktail (Nacalai Tesque). Cell lysates were boiled for 10 minutes with 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific). Proteins were separated using the NuPAGE SDS gel system (Thermo Fisher Scientific), electroblotted onto a PVDF membrane (Amersham Hybond‐P; Cytiva) and subjected to immunodetection using the following primary Abs at 1:1000 dilution; monoclonal mouse antibodies: anti‐α‐tubulin B‐5‐1‐2 (T5168; Merck), anti‐Cyclin B (610219; BD Biosciences), and anti‐Bcl‐2 (sc‐509; Santa Cruz Biotechnology); polyclonal rabbit antibodies: anti‐CHAMP1 (HPA008900; Atlas Antibodies), anti‐Mcl‐1 (4572; Cell Signaling Technology), anti‐Bcl‐xL (2762; Cell Signaling Technology), anti‐MAD2L2 (12683‐1‐AP; Proteintech), and anti‐Bak (3814; Cell Signaling Technology); and monoclonal rabbit antibody: anti‐Bim (2933; Cell Signaling Technology). Blocking and Ab incubations were carried out in 3% nonfat dry milk. Proteins were visualized using HRP‐labelled secondary Abs (1:5000; Santa Cruz Biotechnology) and enhanced chemiluminescence using ECL Prime Western Blotting Detection Reagents (Cytiva), according to the manufacturer’s instructions.
3
Western Blot Analysis of Apoptosis Regulators
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Western blot was performed as previously described [19 (link)]. Primary antibodies used were anti-BIM (1:1000, #2933 (C34C5) Cell Signaling Technology, Danvers, MA, USA), anti-MCL-1 (1:1000, #5453 (D35A5) Cell Signaling Technology), anti-BCL-2 (1:1000, ab32124 (E17), Abcam, Cambridge, United Kingdom), and anti-BCL-XL (1:1000, #2764 (54H6) Cell Signaling Technology).
4
Protein Expression Analysis in HeLa Cells
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HeLa cells were lysed in 1% NP-40 lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, 1 mM DTT and proteinase inhibitors (halt inhibitor, Thermo #78442), and subjected to separation on 8 or 10% SDS-PAGE gels and Western blot (10 μg whole cell lysate per lane). Antibodies used for Western blot were anti-Bim (Cell Signaling, 2933), anti-Pten (Cell Signaling, 9559), anti-Phlpp2 (Bethyl, A300-661A-1), and anti-Ship1 (Cell Signaling, 2728).
5
Metformin and TRAIL-Induced Apoptosis
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Metformin was purchased from Wako (Richmond, VA, USA). TRAIL (Recombinant human) was purchased from Millipore (Millipore, Darmstadt, Germany.) Protein G PLUS-Agarose, Anti-Bax, anti-Bcl-2, anti-Mcl-1(IP), anti-Ub and anti-Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-phospho ERK, anti-ERK, anti-phospho JAK2, anti-JAK2, anti-phospho AMPK, anti-AMPK, anti-phospho mTOR, anti-mTOR, anti-phospho AKT, anti-AKT, anti-phsopho GSK3β, anti-GSK3β, anti-Noxa, anti-Puma, anti-Bim, anti-phospho Mcl-1, anti-Mcl-1(WB), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-phospho STAT3, anti-STAT3, and anti-PARP-1 were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Anti-Mule antibody was purchased from Abcam (Cat. No. ab70161). For the secondary antibodies, anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP were purchased from Cell Signaling (Beverly, MA, USA).
6
Immunoblotting of Apoptosis Markers
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Cells were lysed with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Total cell lysates were subjected to SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes by using an iBlot 2 Dry Blotting System (Thermo Fisher Scientific). Immunoblotting was carried out using the following antibodies; anti-caspase-3, anti-caspase-8, anti-cleaved caspase-8, anti-BIM, anti-Fas, and anti-β-actin antibodies (Cell Signaling Technology). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), and imaging was performed with a ChemiDoc Touch imaging PC system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were performed at least twice and the representative data of one experiment are shown.
7
Targeting BRAF, AKT, and MEK in Cancer Cells
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The BRAF inhibitor vemurafenib (PLX4032), AKT inhibitor MK2206, and MEK inhibitor U0126 were all obtained from Selleck Chemicals (Houston, TX, USA). The reactive oxygen species (ROS) inhibitor NAC (N-acetyl-l -cysteine) was purchased from Beyotime (Shanghai, China). PLX4032 and U0126 were both dissolved in dimethylsulfoxide (DMSO) in 50 mM stock. MK22062 was dissolved in DMSO in 20 mM stock. NAC was dissolved in water in 50 mM stock. Primary antibodies were used as follows: anti-VCAM-1 was obtained from Abcam (Cambridge, UK), anti-ERK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-AKT, anti-phospho-AKT (Ser473), anti-mTOR, anti-phospho-mammalian target of rapamycin (mTOR), anti-cleaved caspase-3, anti-cleaved poly (ADP-ribose) polymerase (PARP), anti-Bim, anti-Bcl-xl, anti-Mcl-1, anti-Vimentin, anti-Snail, anti-ATP-binding cassette sub-family G member 2 (ABCG2), anti-CD44, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies were all purchased from Cell Signaling Technology (Beverly, MA, USA).
8
TRAIL-induced Apoptosis Regulation Mechanisms
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Cannabidiol and VAS2870 were purchased from Sigma (St. Louis, MO, USA). TRAIL and anti-DR5 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-Bak, anti-Bcl-2, anti-Mcl-1, anti-Bcl-xL, and anti-DR4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-BIM, anti-survivin, anti-Bid, anti-IRE1α, anti-phospho-IRE1α, anti-Bip, anti-GRP94, anti-ATF6, anti-eIF2α, anti-phospho-eIF2α, anti-CHOP, anti-cleaved PARP, anti-caspase-3, and anti-caspase-9 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). For the secondary antibodies, anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG HRP were purchased from Cell Signaling Technology.
9
Comprehensive Protein Expression Analysis
10
Comprehensive Protein Extraction and Analysis
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Cellular proteins were extracted in lysis buffer (iNtRON Biotechnology, Seongnam, Korea) and quantified using BCA protein assay kits (Pierce, Rockford, IL, USA). Cell lysates were run on a 10% SDS–polyacrylamide gel and were transferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA). The membrane was blocked in 5% nonfat dry milk in PBS-Tween-20 (0.1%, v/v) for 1 h and incubated with primary antibodies overnight at 4 °C. After washing with TBS containing 0.1% Tween-20, the membrane was incubated for 1 h with secondary antibodies. Alliance-Mini. An HD9 chemiluminescence documentation system (UVItec Cambridge, UK) was used to visualize target proteins. Anti-LGR5 (ab238518) and anti–GAPDH (catalog number: #TA505454) antibodies were purchased from Abcam and Origene, respectively. Anti-ERK (catalog number: #4695), anti-AKT (catalog number: #4691), anti-phospho-ERK (catalog number: #4370), anti-phospho-AKT (catalog number: #4060), anti-cleaved PARP (catalog number: #5625), anti-cleaved caspase-3 (catalog number: #9661) and anti-BIM (catalog number: #2933) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
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