Tissues and cells were lysed with RIPA lysis buffer supplemented with 1% protein phosphatase inhibitor mixture, and the sample was then centrifuged (12,000 rpm) at 4°C for 15 min to get the protein supernatant. The concentration of total protein was detected using the BCA assay kit (Thermo). After separation in a 10–12% SDS-PAGE gel, the proteins were then transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked for 1.5 h with 5% (w/v) non-fat milk dissolved in tris-buffer saline solution containing 0.1% (v/v) Tween-20 (TBST) at RT and further incubated overnight at 4°C with the following primary antibodies: ZO-1 (1:1,000), P120-catenin (1:1,000), β-catenin (1:1,000), occludin (1:1,000), claudin-5 (1:1,000), ATG5 (1:1,000), Beclin1 (1:1,000), AKT (1:1,000), p-AKT (1:1,000), FOXO1 (1:1,000), KLF4 (1:1,000), β-actin (1:10,000), and LC3II (1:1,000; Abcam). Next, after washing three times with TBST, the membrane was submerged in a corresponding HRP-conjugated secondary antibody for 1 h at RT. Signals of protein expression were collected by a ChemiDoc XRS imaging system (Bio-Rad). The relative density of the band was quantified by ImageJ software. All of the primary antibodies (except for anti-LC3II) were purchased from Proteintech.
P120 catenin
Manufactured by Abcam
Sourced in United States
P120-catenin is a member of the catenin protein family. It plays a role in the regulation of cell-cell adhesion and is involved in the modulation of cadherin-mediated cell-cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by Abcam (3 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
β actin, by Abcam (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Bcl 2, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Foxo1, by Abcam (1 mentions)
Lc3 2, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Anti lc3ii, by Proteintech (1 mentions)
E selectin, by Abcam (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Icam 1, by Abcam (1 mentions)
Vcam 1, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Cleaved parp1, by Abcam (1 mentions)
Connexin 43, by Thermo Fisher Scientific (1 mentions)
α catenin, by Abcam (1 mentions)
6 protocols using p120 catenin
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Apoptotic and Cell-Cell Adhesion Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysis Buffer and BCA Protein Assay Kit from Thermo Fisher were employed to extract total protein and determine their concentrations, respectively. After separation using 10% SDS-PAGE, protein bands were transferred to the PVDF membrane. After 1 h of blocking with 5% non-fat milk, samples were incubated overnight with primary antibodies against BAX (1:1000, Abcam, Boston, MA, USA), BCL2 (1:1000, Abcam, Boston, MA, USA), Caspase3 (1:1000, Abcam, Boston, MA, USA), cleaved PARP1 (1:1000, Abcam, Boston, MA, USA), E-selectin (1:1000, Abcam, Boston, MA, USA), ICAM1 (1:1000, Abcam, Boston, MA, USA), VCAM1 (1:1000, Abcam, Boston, MA, USA), P120-catenin (1:1000, Abcam, Boston, MA, USA), β-catenin (1:1000, Abcam, Boston, MA, USA), and E-cadherin (1:1000, Abcam, Boston, MA, USA) at 4° C. Finally, samples were incubated for 1 h with HRP-conjugated secondary antibodies and the protein bands were analyzed.
3
Antibody Characterization for Cell Signaling
4
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animal tissues and cells were lysed with RIPA buffer (pH 7.4, 50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and EDTA). Tissue homogenates were centrifuged at 12 000 rpm, for 15 minutes at 4°C. The extracted supernatant was quantified by the BCA assay (Thermo). Total protein (40 μg) was separated on a 12% gel and transferred onto a PVDF membrane (Bio‐Rad). The membranes were blocked for 1.5 hours in 5% dry milk dissolved in 0.1% Tween‐20 in TBS at room temperature and incubated overnight with the following primary antibodies: P120‐catenin (1:1000, Abcam), β‐catenin (1:1000, Abcam), occludin (1:1000, Abcam), cleaved caspase‐3 (1:1000, Abcam), Bcl‐2 (1:1000, Abcam), Bax (1:1000, Abcam), Tomm20 (1:1000, Abcam), ATG7(1:1000, Novus), beclin1 (1:1000, Abcam), LC3II (1:1000, Novus) and GAPDH (1:10 000, Yeasen). Then, the membrane was washed three times with TBST and incubated with a horseradish peroxidase conjugated secondary antibody. A ChemiDoc™ XRS imaging system (Bio‐Rad) was used to visualize the signals. Quantity one was used to analyse the relative density of the bands, and band density was normalized to that of GAPDH. All experiments were repeated at least in triplicate.
5
Endothelial Cell Culture and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMSO (Sigma-Aldrich) was used to dissolve non-water soluble reagents and as a vehicle control. Endothelial Cell Medium (ECM) which contains 500 ml of basal medium, 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of endothelial cell growth supplement (ECGS, Cat. No. 1052) and 5 ml of penicillin/streptomycin solution (P/S, Cat. No. 0503) were purchased from ScienCell Research Laboratories. Antibodies against Occludin, Claudin5, CHOP, GRP78, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p62 and microtubule-associated protein-1 light-chain 3 (LC3) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-β-catenin, P120-catenin, cleaved caspase-12, and PDI were purchased from Abcam. All chemicals including RA were from Sigma Chemical Company.
6
Protein Expression Profiling in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of proteins (20 μg) lysed from cells were resolved in SDS-PAGE gel and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). Primary antibodies against MUC16 (1:10000, Abcam, Cambridge, UK), p120-catenin (1:1000, Abcam, Cambridge, UK), FLAG (1:500, Proteintech Group, Inc., IL, USA), GAPDH (1:1000, Cell Signaling Technology, Beverly, MA), β-actin (1:1000, Abcam, Cambridge, UK), lamin B1 (1:500, Abcam, Cambridge, UK), Rac1 (1:1000, Abcam, Cambridge, UK), Cdc42 (1:125, Abcam, Cambridge, UK), and RhoA (1:5000, Abcam, Cambridge, UK) were used. The synthetic peptide of primary antibodies against MUC16 corresponding to Human MUC16 aa 12,450–12,550. Anti-rabbit or anti-mouse IgG (1:2000, Cell Signaling Technology, Beverly, MA) was used as the secondary antibody. For subcellular fractions, cytoplasmic or nuclear proteins were extracted using a cytoplasmic protein extraction kit or a nuclear protein extraction kit (SAB, MD, USA). The purity of subcellular fractions was confirmed using antibodies against GAPDH (a cytoplasmic marker) and lamin B1 (a nuclear marker).
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!