Membrane‐associated RING‐CH‐1 (Bioss, bs‐9335, Beijing, China), Phospho‐AKT Ser473 (SAB, 11054, Randallstown, MD, USA), total AKT (SAB, 21054), GAPDH (Proteintech, 10494‐1‐AP, Wuhan, Hubei, China), PI3K p110 β (Proteintech, 20584‐1‐AP), β‐catenin (Proteintech, 51067‐2‐AP), Mcl‐1 (Proteintech, 16225‐1‐AP), Bcl‐2 (Proteintech, 12789‐1‐AP), Cleaved caspase‐3 (CST, 9661, Fall River, MA, USA), Cleaved caspase‐7 (CST, 8438), secondary antibodies (Peroxidase‐conjugated Goat anti‐Rabbit IgG; ZSGB‐BIO, ZB‐2301, Beijing, China), Caspase‐3/7 Inhibitor I (ApexBio,A1925, Houston, TX, USA) and Pirarubicin (Selleck, Houston, TX, USA) were obtained commercially.
Mcl 1
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
Mcl-1 is a lab equipment product from Proteintech. It is a protein that plays a key role in regulating apoptosis, or programmed cell death, in cells.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Proteintech (6 mentions)
Bcl2 associated x (bax), by Proteintech (4 mentions)
Gapdh, by Proteintech (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Caspase 8, by Cell Signaling Technology (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
β actin, by Proteintech (2 mentions)
Caspase 3 7 inhibitor 1, by Apexbio (1 mentions)
Pirarubicin, by Selleck Chemicals (1 mentions)
β catenin, by Proteintech (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by ZSGB-BIO (1 mentions)
Microtubule associated protein 1 light chain 3, by Merck Group (1 mentions)
P70s6k, by Merck Group (1 mentions)
Peroxidase linked anti mouse antibody, by Merck Group (1 mentions)
18 protocols using mcl 1
1
Protein Expression and Apoptosis Analysis
2
Western Blot Analysis of Apoptosis and Autophagy
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For western blots, 2 × 105 OVCAR-3 or A2780 cells per well were plated in a 6-well plate for 24 h. Subsequently, the cells were incubated with the appropriate drug for distinct time periods and the western blots were performed as previously described [36 (link)] using the following antibodies: Capsese-3 (Cell Signaling Technology, USA, #9661), PARP (Cell Signaling Technology, USA, #9542), Microtubule-associated protein 1 light chain 3 (LC3) (Sigma, USA, #L7543), p62/SQSTM1/SQSTM1 (Cell Signaling Technology, USA, #5114), ATG5 (Cell Signaling Technology, USA, #9980), ATG7 (Cell Signaling Technology, USA, #8558), p-AKT (Cell Signaling Technology, USA, #9271), AKT (Cell Signaling Technology, USA, #9272), BAX (Proteintech Group, USA, #23931-1-AP), Bcl-2 (Proteintech Group, USA, #12789-1-AP), and Mcl-1 (Proteintech Group, USA, #16225-1-AP), p-p70S6K (Thr389) (Sigma, USA, #MABS82), p70S6K (Sigma, USA, #06-926), p-mTOR (S2448) (Abcam, USA, #ab109268), -S6 Ribosomal (Cell Signaling Technology, USA, #5364), S6 Ribosomal (Cell Signaling Technology, USA, #2317), mTOR (Abcam, USA, #ab2732), and β-actin (Origene, USA, #TA811000), peroxidase-linked anti-rabbit antibody (Cell Signaling Technology, Danvers, USA, #7074) or peroxidase-linked anti-mouse antibody (Sigma-Aldrich, St. Louis, USA, #A9044).
3
Sinomenine Modulates STAT3 and AMPK
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Sinomenine was purchased from Solarbio Life Sciences (#SS8560,Solarbio, Beijing, China), dissolved in DMSO and placed away from light at −20°C. Anti-MARCH1 antibody (#bs-9335R) was obtained from Bioss (Beijing, China). Anti-p-STAT3 (#ab32143), p-AMPK (#ab92701) and AMPK (#ab32047) antibodies were purchased from Abcam (Cambridge, United Kingdom). Anti-Bcl-2 (#12789-1-AP), CyclinB1 (#55004-1-AP), CyclinD1 (#26939-1-AP), Mcl-1 (#16225-1-AP), Bax (#50599-2-Ig), STAT3 (#10253-2-AP), GAPDH (#10494-1-AP) and Peroxidase-conjugated Affinipure Goat anti-Rabbit IgG (H + L) (#SA00001-2) antibodies were purchased from the Proteintech Group (Chicago, IL, United States).
4
Autophagy Regulation in Cell Death
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Cell culture materials were obtained from Invitrogen and fetal calf serum was from Gibco. 3-bromopyruvate, chloroquine diphosphate, 3-methyladenine (3-MA), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Nec1 were purchased from Sigma-Aldrich. z-VAD-fmk was purchased from Calbiochem. JC-1 and Annexin V-FICT/PI assay were purchased from KeyGEN BioTECH (China). GFP-LC3 plasmid was purchased from GeneCopoeia. The following antibodies were used: LC3, Bclin-1 (MBL), Bax, Bak, Bcl-2, Mcl-1 (ProteinTech), Atg7 (Beyotime), RIPK1 (Santa cruz), RIPK3 (Cell Signaling).
5
Nuclear Protein Extraction and Western Blot Protocol
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Nuclear protein was extracted according to the manufacturer's instruction of the nuclear and cytoplasmic protein extraction kit (Beyotime). The protocol for western blot has been described in detail in our previous publication.56 (link) Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 (Epitomics, Burlingame, CA, USA); STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 (Cell Signaling Technology); antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 (Proteintech, Wuhan, Hubei, China); GAPDH (Beyotime).
6
Western Blot Analysis of Apoptosis Markers
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Cells lines were lysed using 1× RIPA Lysis (Solarbio, China) containing PMSF (1:100, Solarbio, China). Protein samples were quantified using the BCA protein assay kit (Solarbio, China) as manufacturer’s instructions. We loaded 20 μg protein per well into 10–12% gels and then transferred it to PVDF membranes using the Trans-Blot Turbo transfer system (Bio-Rad) according to the manufacturer’s instructions. Membranes were blocked for 1 h in 5% BSA and stained with primary antibody overnight at 4 °C. The following primary antibodies were tested: Bcl-xL (1:1000, Abcam), Bcl-xS (1:1000, GeneTex), SQSTM1 (1:1000, Cell Signaling), LC3 (1:1000, Abcam), Beclin-1 (1:1000, Abcam), Cleaved-Caspase3 (1:1000, Cell Signaling), Caspase3 (1:1000, Cell Signaling), Cytochrome c (1:1000, Immunoway). BCL2 (1:1000, Proteintech), MCL1 (1:1000, Proteintech). After washing the blots three times for 10 min each with TBST, the secondary antibody anti-rabbit horseradish peroxidase (1:10000, Immunoway) or anti-mouse horseradish peroxidase (1:10000, Immunoway) was added for 1 h at room temperature. Following another round of 3 × 10 min washes, the membranes were imaged on the Alpha Innotech followed by quantification using Image J.
7
Apoptosis Signaling Pathway Analysis
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Standard western blot procedures were used on whole cell lysates and probed with antibodies specific to MCL-1 (Proteintech, UK), ACTIN (Sigma, UK), PARP (Cell Signaling, UK), BAK (Cell Signaling, UK), BAX (Santa Cruz, CA, USA), HSP70 (Cell Signaling, UK), Active Caspase 3 (Cell Signaling, UK).
8
Quantitative Immunoblot Analysis of BCL-2, MCL-1, and MYC
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Cell lysates were prepared as described previously [26 (link)] and fractionated in 7.5–15% SDS-PAGE gradient electrophoresis gel. Proteins were transferred on PVDF membrane and specific proteins were detected using antibodies against BCL-2 (Cell Signalling Technology, Danvers, MA, USA), MCL-1 (Proteintech, Rosemont, IL, USA), MYC (Novus Biologicals, Centennial, CO, USA) and β-tubulin (Abcam, Cambridge, UK). β-tubulin was used as a loading control. Chemiluminescent detection was carried out using WesternBright ECL (Advansta, San Jose, CA, USA) on ChemiDoc™ XRS+ System (BIORAD, Hercules, CA, USA). Quantitative evaluation was performed using ImageJ software (1.48v).
9
Neutrophil Lysis and Immunoblotting
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Neutrophils were lysed for 30 min in ice‐cold lysis buffer with 4 mM Diisopropyl fluorophosphate (Armstrong et al., 2016 (link)). Unless noted, 10–20 μg/μl total cell lysates were separated by 12% SDS‐PAGE and immunoblotted with antibodies for MCL‐1 (1:500, Proteintech, Rosemont, IL, USA), XIAP, caspase 8, caspase 9, caspase 3 and β‐actin (1:1000, Cell Signaling, Danvers, MA, USA). Secondary antibodies were used at 1:2000 dilution (Cell Signaling, Danvers, MA, USA). The ECL system (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) was used to visualize antigen–antibody reactions. Densitometric values were calculated using Image Lab software (BioRad, Hercules, CA, USA).
10
Western Blot Analysis of MCL-1, PCNA, and GAPDH
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After NOZ and SGC-996 cells transfection at 48 h, cells were collected and lysed in RIPA lysis buffer (Beyotime, Shanghai, China). After the protein was qualitied by using BCA protein assay kit (Beyotime, Shanghai, China), equal amounts of proteins (30 μg) were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the PVDF membranes (Millipore, Billerica, MA, USA). Following blocked the non-specific binding sites with 5% non-fat milk, the membranes were with antibodies against MCL-1 (1:500, Protein tech, Wuhan, Hubei, China), PCNA (1:2000, Abcam, Cambridge, MA, USA), or GAPDH (1:2000, Abcam, Cambridge, MA, USA) overnight at 4 °C, along with horseradish peroxidase-labeled IgG for 2 h at room temperature. The bands were visualized using Image Lab after the addition of luminol-based chemiluminescent substrate (Pierce, Rockford, IL, USA). The results were analyzed using Image Lab software (NIH, Bethesda, MD, USA).
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