Proteins were separated and detected as previously described [28 (link)]. In brief, SDS-PAGE gel (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) was used to separate proteins and then transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Blocking of membranes was achieved by using 5% dry milk dissolved in Tris Buffer Saline with 1% Tween 20 (TBST) and the following antibodies were incubated overnight at 4 °C: rabbit anti-BCL-2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit anti-NOXA (CST14766, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-phospho-ERK1/2 (CST4376, Cell Signaling), rabbit anti-Actin (CST4970, Cell Signaling). Anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling) was used and immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad). When required, immunoblots were stripped in 0.1 M glycine pH 2,5, 2% SDS for 40 min and washed in TBS. The visualization of the bands was done using the LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to quantify the integrated optical density of bands.
Rabbit anti mcl 1
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-Mcl-1 is a primary antibody that specifically recognizes the Mcl-1 protein. Mcl-1 is a member of the Bcl-2 family of proteins and plays a role in the regulation of apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti bcl 2, by Cell Signaling Technology (3 mentions)
Rabbit anti bcl xl, by Cell Signaling Technology (2 mentions)
Rabbit anti bim, by Cell Signaling Technology (2 mentions)
Rabbit anti survivin, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel 12, by Bio-Rad (1 mentions)
Las4000 imager, by GE Healthcare (1 mentions)
Rabbit anti actin, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Hybond, by Cytiva (1 mentions)
Mouse anti β actin antibody, by Cell Signaling Technology (1 mentions)
Mouse anti stat3, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Rabbit anti pstat3, by Cell Signaling Technology (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Anti rabbit igg h l antibody, by Cell Signaling Technology (1 mentions)
Bt474 cells, by American Type Culture Collection (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Rabbit anti bcl 2, by Abcam (1 mentions)
Precast polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
9 protocols using rabbit anti mcl 1
1
Quantitative Immunoblotting of Apoptosis Regulators
2
Western Blot Analysis of IL-6 Signaling
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U266 or 8266 cells were harvested 24 or 48 hours after IL-6 treatment and washed with cold PBS. Total proteins extracted from the cells using a RIPA cell lysis buffer (Wolsen, China) were separated on 10% SDS polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membrane (Millipore, USA). The membranes were treated with mouse anti-STAT3, rabbit anti-MCL-1, anti-PI3K or anti-MAPK1/2 antibodies, rabbit anti-pSTAT-3, anti-pMAPK1/2 antibodies, and mouse anti-β-actin antibodies (1:1000 dilution) (Cell Signaling Technology, USA). The membranes were then reacted with the HRP-conjugated secondary antibodies before subjected to enhanced chemiluminescent (ECL) detection on an ECL machine (Pierce, USA). The blots were scanned and the band density was measured on the Quantity One imaging software.
3
Paraformaldehyde Fixation and Immunohistochemistry
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The tissues samples were fixed using 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and divided into 4 µm slices. The tissues samples were dehydrated with xylene and ethanol (100% ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min and 70% ethanol for 5 min; Nanjing Chemical Reagent Co., Ltd., Nanjing, China). Subsequently, the sections were incubated in a blocking solution of 10% normal goat serum and with rabbit anti-Mcl-1 (dilution, 1:200; catalog no. 94296; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight and subsequently incubated with anti-rabbit IgG (H+L) antibody (dilution, 1:500; catalog no. 8889; Cell Signaling Technology, Inc.) for 1 h at room temperature. The sections were incubated with streptavidin-biotin complex conjugated to horseradish peroxidase and diaminobenzene for 10 min at room temperature. Tissue samples were observed using a fluorescence microscope (LSM710; magnification, ×20; Carl Zeiss Microscopy GmbH, Jena, Germany).
4
Immunoprecipitation and Western Blot Analysis of Apoptosis Regulators
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Proteins were extracted with cold EB buffer (50 mmol/L Hepes pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 5 mmol/L EDTA, 5 mmol/L EGTA) in the presence of phosphatase and protease inhibitors (Thermo Fisher Scientific). Immunoprecipitations were performed by incubating protein extracts with either the anti-BCL-XL or the anti-MCL1 primary antibody and Protein A sepharose beads (GE Healthcare) for 1 hour at 4°C. Immunoprecipitated or total proteins were electrophoresed on precast polyacrylamide gels (Invitrogen) and transferred onto nitrocellulose membranes using a Trans-Blot Turbo Blotting System (Bio-Rad). Membrane-bound antibodies were detected by the enhanced chemiluminescence system (Promega). Primary antibodies were the following: rabbit anti-BCL2 (Abcam, #ab32124), rabbit anti-BCL-XL (Cell Signaling Technology, #2762), rabbit anti-MCL1 (Cell Signaling Technology, #94296), rabbit anti-BIM (Cell Signaling Technology, #2933), and mouse anti-vinculin (Sigma-Aldrich, V9131). BT-474 cells were purchased from ATCC and used as controls for protein expression.
5
Western Blotting and Immunostaining Antibodies
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Primary antibodies used in western blotting and immunostaining were as follows: mouse anti-cytochrome c (BD Biosciences, 556433); rabbit anti-phosphorylated H2A.X (Ser139) (Cell Signaling Technology, 9718); rabbit anti-human GAPDH (Trevigen, 2275-PC-100); rabbit anti-cleaved PARP (Cell Signaling Technology, 5625), rabbit anti-phospho-p53 (Cell Signaling Technology, 9284), mouse anti-TP53/p53 (Santa Cruz Biotechnology, SC-126), mouse anti-p21 Waf1/Cip1 (Cell Signaling Technology, 2946), rabbit anti-LC3 (MBL International, PM036), rabbit anti-SQSTM1/p62 (Cell Signaling Technology, 5114), anti-ATG3 (MBL International, M133-3), rabbit anti-Mcl-1 (Cell Signaling Technology, 5453), rabbit anti-phospho-histone H3 (Ser 10) (Cell Signaling Technology, 9701), rabbit anti-phospho-histone H2A.X (Ser 139) (20E3) (Cell Signaling Technology, 9718), mouse anti-cyclin B1 (Cell Signaling Technology, 4135). Horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology.
6
Regulation of Survivin and Cell Cycle Proteins
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Sources were: YM155 (Selleck Chemicals); rabbit anti-Survivin IgG (#AF886) (R&D Systems); rabbit anti-P-Rb (S807/811, #9308), rabbit anti-P-Cyclin D1 (T286, #3300), mouse anti-Cyclin D1 (#2926), rabbit anti-Mcl-1(#5453), mouse anti-Cyclin D3 (#2936), rabbit anti-P-Akt (T308, #6955), rabbit anti-Akt (S473, #9271), rabbit anti-rS6 (#2217), rabbit anti-P-rS6 (S235/236, #2211), rabbit anti-P-p70S6K1 (T389, #9205), rabbit anti-mTOR (#2972), rabbit anti-P-mTOR (S2448, #2971), rabbit anti-Raptor (#4978, #4972) rabbit anti-P-Raptor (S792, #2083) rabbit anti-Rictor (#2140), rabbit anti-P-Rictor (T1135, #3806), rabbit anti-AMPKα (#5831), rabbit anti-P-AMPKα (T172, #2535) rabbit anti-AMPKα1 (#2795), rabbit anti-AMPKα2 (#2757), rabbit anti-4E-BP1 (#9644), rabbit anti-P-4E-BP1 (S65) (Cell Signaling Technologies); rabbit anti-Rb (sc-50), rabbit anti-Cyclin D1 (sc-753), rabbit anti-Cyclin D2 (sc-593), mouse anti-GAPDH (sc-51907), mouse anti-α-tubulin (sc-5386), mouse anti-P-rS6 (S240, sc-293143), mouse anti-rS6 (sc-74459), mouse anti-p70S6Kα (sc-39367) (Santa Cruz Biotechnologies, Inc.); mouse anti-β-actin (#A-5441), cycloheximide (CHX), actinomycin D (Act D), MG132, curcumin, (Sigma-Aldrich, Inc.); calyculin A, rapamycin and Compound C (LC labs, Inc.) DMEM/F12 (Media Tech); characterized fetal bovine serum (FBS) (HyClone).
7
Immunoblotting for STAT3 Signaling
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Rabbit anti-phosphorylated (anti-phospho)-STAT3 at tyrosine 705 (Tyr705) and serine 727 (Ser727), rabbit anti-STAT3, rabbit anti-survivin, rabbit anti-Bcl-2, rabbit anti-Mcl-1, rabbit anti-β-actin, and anti-rabbit HRP-conjugated IgG were purchased from Cell Signaling Technology. Anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgG was purchased from Santa Cruz Biotechnology (Dallas, TX, US).
8
Protein Expression Analysis by Western Blot
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Cells were seeded into 6-well dishes, treated with 20 µM Bpep or Dpep for 72 h and processed for Western immunoblotting and imaging as previously described [33 (link)]. Antibodies used were: rabbit anti-survivin (Cell Signaling Technology, Danvers, MA, USA #2808), rabbit anti-MCL-1 (Cell Signaling Technology, Danvers, MA, USA #94296s), rabbit anti-BCL-2 (Cell Signaling Technology, Danvers, MA, USA #15071s) and mouse anti-ACTIN (Cell Signaling Technology, Danvers, MA, USA #3700).
9
Investigating Protein-Protein Interactions via Co-IP
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Co-Immunoprecipitation (co-IP) was performed with 3-day post-infection (95–100%) THP-1 cells using Pierce Protein A/G Agarose kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Protein samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and blocked for 1 h at room temperature in Tris-buffered saline with 5% nonfat dry milk and 1% Triton-X 100. Primary antibodies included mouse anti-FBW7 (1:1000; R&D Systems, used to detect both unmodified and ubiquitinated FBW7), rabbit anti-TRP120 peptide antisera (1:10,000), rabbit anti-GAPDH (1:10,000; Proteintech, Rosemont, IL), rabbit anti-NICD (1:1000; Cell Signaling Technology, Danvers, MA), rabbit anti-p-c-Jun (1:1000; Cell Signaling Technology), rabbit anti-MCL1 (1:1000; Cell Signaling Technology), rabbit anti-K48 linkage-HRP conjugated (1:1000; Cell Signaling Technology), mouse anti-FK2 (1:500; Cell Signaling Technology), and mouse anti-cMYC (9E10) (1:500, Santa Cruz Biotechnology, Dallas, TX). Secondary antibodies included horseradish peroxidase-labeled goat anti-rabbit IgG and anti-mouse IgG (1:20,000; Kirkegaard & Perry, Gaithersburg, MD). Densitometry was performed using ImageJ software.
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