All the protein was collected by using RIPA lysis buffer (Beyotime Biotechnology, P0013, Shanghai, China). Protein was quantified using Coomassie blue staining in microplate spectrophotometer (Thermo Scientific, Multiskan MK3, Waltham, MA, USA) at 594 nm. Identical amount of protein was separated into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then the gels were transferred onto nitrocellulose membrane (GE Healthcare Life Sciences, Amersham™ Protran™ 0.45 NC, Chicago, IL, USA) after electrophoresis. Membranes were blocked by 5% skim milk for 2 hrs. Then, the membranes were incubated with primary antibodies (E-cadherin (ECAD), N-cadherin (NCAD), Vimentin (VIM), and β-actin were purchased from Proteintech group, Wuhan, China; PKMYT1, AKT, mTOR, S6, Phospho-AKT, Phospho-mTOR, Phospho-S6, and Twist1 were purchased from Cell Signaling Technology, Danvers, MA, USA) in 5% bovine serum albumin overnight at 4°C. After rewarming to room temperature for 1 hr, the membranes were incubated with secondary antibody for 1 hrat 37°C. We used enhanced chemiluminescence (Beyotime Biotechnology, Shanghai, China) to detect the blots with ChemiDoc MP (Bio-Rad Laboratories, ChemiDoc MP, Hercules, CA, USA) and the images were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Pkmyt1
Manufactured by Cell Signaling Technology
Sourced in China
PKMYT1 is a protein kinase that plays a role in regulating cell cycle progression. It is involved in the phosphorylation of proteins that control G2/M phase transition and mitotic entry.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Multiskan mk3, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Twist1, by Cell Signaling Technology (1 mentions)
Amersham protran 0.45 nc, by GE Healthcare (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Protran, by Cytiva (1 mentions)
Thymidine, by Merck Group (1 mentions)
Ro 3306, by Merck Group (1 mentions)
Myc tag, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Azd1775, by Selleck Chemicals (1 mentions)
P cdk1 tyr15, by Cell Signaling Technology (1 mentions)
3 protocols using pkmyt1
1
Western Blot Analysis of EMT and PI3K/AKT Pathway
2
Investigating DNA Damage Response Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ODF2L (catalog 23887-1-AP) was purchased from Proteintech. PKMYT1 (catalog A302-424A, Thermo Fisher Scientific, for immunoprecipitation) and PKMYT1 (catalog H00009088-B01P, Abnova, for immunoblotting) antibodies were used for immunoprecipitation followed by immunoblotting. In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology. Alexa Fluor 594–conjugated anti–p-histone γH2AX S139 (Ser139) antibody was purchased from BioLegend (catalog 613410). Alexa Fluor 488–conjugated anti–p-histone γH2AX (Ser139) antibody was obtained from MilliporeSigma (catalog 05-636-AF488). The p-PP1Cα T320 (catalog ab62334) antibody was purchased from Abcam. Antibodies against FLAG (catalog F1804/M2, F7425); glutathione S-transferase (GST) (catalog G1160/GST-2); Myc tag (catalog 05-419); and β-actin (catalog A1978/AC-15) were obtained from MilliporeSigma. AZD1775 (Selleckchem; catalog S1525); Ro-3306 (MilliporeSigma; catalog SML0569); and thymidine (MilliporeSigma; catalog T1895) were prepared as 10 mmol/L solutions in DMSO.
3
Immunofluorescence Staining of Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After PBS washing, slides underwent antigen retrieval through low pressure cooking in 10 mM Tris/EDTA pH 9.0/0.5% Tween-20 for 3 minutes. Sections were permeabilized with PBS/10% Methanol for 10 minutes. Slides were blocked in PBS containing 3% Fetal bovine serum for 1 hour. Slides were incubated with antibodies (1:100) to: SFRP1 (Cell Signaling Technology, cat no. 3534), SFRP2 (Santa Cruz Biotechnology, cat no. sc365524), PKMYT1 (Cell Signaling Technology, cat no.4282), TGF-α (Santa Cruz Biotechnology, cat no. 374433), Collagen IVα1 (Novus Biologicals, cat no. NB1206586, PDGFR-α (Cell Signaling Technology, cat no.5241), α-SMA (Spring Biosciences, cat no. M4712) or CD34 (Novus Biologicals, cat no. NBP2-32932) overnight in PBS/3%FBS. SRFP1 was detected by donkey anti-rabbit-Alexa-Fluor-647 (Invitrogen, cat no. A31573). SFRP2 and PKMYT1, TGF-α were detected by anti-mouse-Alexa-Fluor-568 (Invitrogen, cat no. A10037). PDGFR-α, Collagen A4, and α-SMA were detected by anti-rabbit-biotinylated antibodies. CD34 was detected by anti-mouse-biotinylated antibodies. This was followed by incubation with streptavidin-alexa-fluor-488 (Invitrogen, cat no. S11223) for 30 minutes. After washing in PBS, slides were counterstained with DAPI and mounted with 1:1: PBS: glycerol.
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!