Cells were harvested 12 h after doxycycline treatment and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Quartett) and a phosphatase inhibitor cocktail (Sigma). For BIM detection, the pan-caspase inhibitor Z-VAD-FMK (MedChemExpress) was added 30 min prior to the addition of doxycycline. The cell lysates were then incubated for 1 h on ice and centrifuged at 15,000g for 30 min at 4 °C. Total protein concentrations for the whole-cell lysates were measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of the lysates were resolved on SDS-PAGE gels and transferred to PVDF membranes. These were then incubated with primary antibody overnight at 4 °C before being washed three times with TBS-T buffer and incubated with a secondary antibody for 1 h at room temperature. Chemiluminescence was generated with ECL Western Blotting Substrate (Thermo Fisher Scientific) and detected using a ChemiDoc MP (Bio-Rad). All images were analyzed using Image Lab (Bio-Rad). The antibodies used for the western blots were: anti-FLAG (Sigma, F1804), anti-BCL-xL (Abcam, ab32370), anti-MCL-1 (Abcam, ab32087), anti-γ-H2A.X (Abcam, ab81299), anti-PARP (Abcam, 191217), anti-β-actin (Abcam, ab6276), HRP-conjugated anti-rabbit IgG (Abcam, ab205718), and HRP-conjugated anti-mouse IgG (Abcam, ab6728).
Anti mcl 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Mcl-1 is a lab equipment product that targets the Mcl-1 protein. Mcl-1 is an anti-apoptotic protein that plays a role in cell survival. Anti-Mcl-1 can be used to study the function and regulation of Mcl-1 in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl xl, by Abcam (6 mentions)
Anti bcl 2, by Abcam (6 mentions)
Anti β actin, by Abcam (3 mentions)
Anti survivin, by Abcam (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti bax, by Abcam (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti puma, by Santa Cruz Biotechnology (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti cc3, by Cell Signaling Technology (2 mentions)
Anti p62, by Abcam (2 mentions)
Anti phospho rb, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti mouse igg, by Abcam (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Anti parp, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti rabbit igg, by Abcam (1 mentions)
16 protocols using anti mcl 1
1
Doxycycline-Induced Apoptosis Analysis
2
Immunohistochemical Staining of Tumor Samples
3
Chidamide Acetylation and Ubiquitination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chidamide was synthesized by Shenzhen Chipscreen Biosciences Ltd. (Shenzhen, China) and dissolved in dimethyl sulfoxide (DMSO). MG132 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibody anti-Mcl-1 was purchased from Abcam® (Cambridge, MA, USA), anti-Acetylated-Lysine and anti-β-actin were obtained from Cell Signaling Technology (Boston, MA, USA), and anti-ubiquitin was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).
4
Immunoprecipitation of Mcl-1 and USP9X
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using 1% CHAPS lysis buffer (1% CHAPS, 50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 2 mM Na3VO4, and complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, #11836170001)). For the Mcl-1 IP, 1 mg of lysate was incubated with 2 μg rabbit anti-Mcl-1 (Santa Cruz, #sc-819) or normal rabbit IgG (Millipore, #12-370) and 50 μl Protein A magnetic beads (Pierce, #88846) for 3 h at 4°C. For the USP9X IP, 1.5 mg of lysate was incubated with 2 μg mouse anti-USP9X (Abnova, H00008239-M01) or normal mouse IgG (Millipore, #12-371) and 50 μl slurry Protein G magnetic beads (Pierce, #88847) for 3 h at 4°C. Beads were washed 3 times with lysis buffer containing 0.2% CHAPS and incubated in 2x sample buffer for 15 min at room temperature. Eluted proteins were separated from the beads and boiled for 10 min. For the Mcl-1 IP, anti-USP9X Cell Signaling #14898 and anti-Mcl-1 Abcam #ab32087 were used for western blot detection and for the USP9X IP, anti-Mcl-1 Cell Signaling #5453 and anti-USP9X Cell signaling #14898 were used for western blot detection.
5
Immunoblotting Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-JAK2 (Tyr1007/Tyr1008), anti-JAK2, anti-phospho-Stat3 (Tyr705), anti-Stat3, anti-phospho-Chk2 (Thr68) and anti-phospho-p53 (Ser15) antibodies were purchased from Cell Signaling Technology (Boston, USA). Anti-Bax, anti-Bcl-2, anti-Mcl-1, anti-Caspase-3, anti-Caspase-9, anti-Survivin, anti-Cyclin A2, anti-phospho-ATM (Ser1981), anti-ATM, anti-γH2A.X (Ser139) and anti-LMP1 antibodies were purchased from Abcam (Cambridge, UK). Anti-pBad (S136) and anti-Bad were purchased from Bioworld Technology (Minneapolis, USA). Anti-p53 and anti-Zta antibodies were purchased from Santa Cruz Biotechnology (Texas, USA). Anti-β-actin and HRP-conjugated goat anti-mouse/rabbit secondary antibodies were purchased from ComWin Biotech (Beijing, China).
6
Oxidative Stress and Apoptosis Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MTT, dimethyl sulfoxide (DMSO), PI, DAPI and DCFH-DA were purchased from Sigma (St Louis, MO). RIPA buffer and JC-1 staining kit were purchased from Beyotime Biotechnology, China. The following antibodies were used: anti-PUMA, anti-p53, anti-Chk1, anti-Chk2, anti-p-Chk1(Ser345), anti-p-Chk2 (Thr68), anti-ATM, anti-p-ATM (Ser1981), anti-PARP, anti-GAPDH and anti-β-actin (Santa Cruz Technology, Santa Cruz, CA, USA); Anti-NOX1, anti-NOX4, anti-DUOX1 and anti-SOD1, anti-Keap1, anti-Nrf2, anti-HO-1, anti-DNA-PKcs, anti-p-DNA-PKcs (Thr 2609), anti-Caspase-3 and anti-Caspase-9 (Cell Signaling Technology, Danvers, MA, USA); Anti-γ H2AX (Ser139), anti-H2AX and anti-JNK, anti-p-JNK, anti-BCL-XL, anti-MCL-1 and anti-BCL-2 (Abcam). Rhodamine (TRITC) AffiniPure Goat anti-Rabbit IgG was from Santa Cruz Biotechnology; KU-60019, VE-821 and NU7026 were obtained from Selleck (USA). SP600125 were obtained from Calbiochem (La Jolla, CA, USA). Z-VAD-fmk was purchased from R&D Systems (Minneapolis, MN). These inhibitors were dissolved in DMSO and diluted to appropriate concentrations with cell culture media. Mitotracker and MitoSOX red mitochondrial superoxide indicator was purchased from Molecular Probe (USA).
7
Anticancer Pathway Regulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Celastrol and MG-132 were obtained from Calbiochem (Cat #474790; San Diego, CA, USA). PS-341 was obtained from Millennium Pharmaceuticals (Cambridge, MA, USA). Cycloheximide (CHX) was purchased from Beyotime (Jiangsu, China). These reagents were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, MO, USA). The primary antibodies were used: anti-CIP2A (HL1925; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-PP2A, anti-MCL-1 and anti-Actin (ab137849, ab32087, ab8226; Abcam, Cambridge, MA, UK); anti-GSK3β, anti-BCL-2 and anti-BCL-xL (#9336, #15071, #2764; Cell Signaling Technology, Danvers, MA, USA). Enhanced chemiluminescence kit was obtained from Yeasen Technology (36222ES60; Shanghai, China).
8
Protein Extraction and Western Blot Analysis of Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cultured cardiomyocyte cells or tissues using RIPA lysis buffer (CWBio), according to the manufacturer’s instructions. 20 µg protein samples were separated by 10% SDS-PAGE and transferred onto the PVDF membrane (EMD Millipore). Following blocking, the membranes were incubated with specific primary antibodies: Anti-p53, anti-PUMA, anti-cleaved caspase-3 and anti-cleaved caspase-9. All the antibodies were purchased from Cell Signaling Technology (CST, Shanghai, China); anti-Bim (B7929, 1:1000) and anti-Bax (#CNB8429,1:1000) were purchased from Sigma-Aldrich (Shanghai, China); anti- Phospho-FoxO3a (Ser294) (#5465S, 1:1000), anti-Bcl-2 (#ab218123,1:1000), anti-MCL-1(#Ab280453,1:1000) and anti-GAPDH (ab9485, 1:2000) were purchased from Abcam; anti-Noxa (OP180, 1:400) was purchased from Calbiochem. Target protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
9
Evaluating Apoptosis Regulators in PDAC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PDAC cells were treated with co-delivery NP, HCQ NP, or PAL NP for 72 h at equivalent drug concentration (PAL = 2.5 μM, HCQ = 10.8 μM). PANC-1 cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 3 wt% bovine serum albumin (BSA). Primary antibodies included anti-phospho-Rb (1:1000, cat#8516, Cell Signaling Technology), anti-p62 (1:100, cat#ab91526, Abcam), anti-Ki67 (1:500, cat#ab15580, Abcam), anti-CC-3 (1:400, cat#9664, Cell Signaling Technology), anti-Bcl-2 (1:150, cat#ab182858, Abcam), anti-Mcl-1 (1:500, cat#ab32087, Abcam), or anti-Bcl-xL (1:500, cat#ab32370, Abcam) accordingly. Cells were further incubated with secondary antibodies, including Alexa Fluor 594 goat anti-rabbit immunoglobulin G (IgG; 1:500, cat#A11012, Thermo Fisher Scientific) or Alexa Fluor 488 goat anti-rabbit IgG (1:500, cat#A11008, Thermo Fisher Scientific), and counterstained with 4,6-diamidino-2-phenylindole. The immunofluorescence staining was visualized by confocal microscopy. Fluorescence intensity of biomarkers including Ki67, Bcl-xL, Mcl-1, and Bcl-2 was quantified with the ImageJ software (v1.5.2) and normalized to the number of cells as indicated by nuclei stain.
10
Cisplatin and Epoxomicin-Induced Cell Death
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cisplatin and epoxomicin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used: anti-p62, anti-Mcl-1 (Abcam, Cambridge, MA, USA); anti-p53,anti-β-actin, anti-VDAC1, anti-LaminA/C, anti-βtublin, anti-p53, antiBax (Proteintech group, Inc., Rosemont, IL, USA); anti-PORLMT (Cell Signaling Technology, Danvers, MA, USA); anti-Bcl-2 (PTI BIO, Liaoning, China); anti-OXPHOS (Thermo Fisher Scientific, Waltham, MA, USA); All antibodies were diluted at a ratio of 1:1000.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!