Cell lysates were generated with PBS/1% NP40 buffer supplemented with protease inhibitor cocktail (Sigma Cat. No. S8820). Protein sample loading buffer was added to cell lysates and proteins were separated on SDS-PAGE (BioRad Criterion TGX Precast Gel). The following primary antibodies were used for immunoblotting at the indicated dilutions: Cell Signaling Technology: AXIN1 (2087 and 2074; 1:1000), BAP1 (13271; 1:1000), BCL2L2 (2724; 1:1000), LRP6 (2560; 1:1000), PTEN (9552; 1:1000), RICTOR (9476; 1:1000), SIRT1 (2493; 1:1000), TBK1 (3504; 1:1000), TLE3 (4681; 1:1000), LKB1 (3050; 1:1000), SUFU (2520; 1:1000), LRP5 (5731; 1:1000). Bethyl Laboratories: BAP1 (A302-243A-T; 1:1000), RICTOR (A300-459A; 1:1000), SIRT1 (A300-688A; 1:1000), TOP1 (A302-589A and 302-590A; 1:1000), VPS35 (A304-727A; 1:1000). Abcam: TBK1 (40676; 1:1000), PPM1A (14824; 1:1000), SUFU (52913; 1:1000). Santa Cruz Biotechnologies: LRP6 (sc-25317; 1:1000), TLE3 (sc-9124; 1:1000), LKB1 (sc-374334; 1:1000), LRP5 (sc-390267; 1:1000). LS BioScience: BCL2L2 (LS-C382259-100; 1:1000), PPM1A (LS-C169090-100; 1:1000). Sigma-Aldrich: CTNNB1 (C2206; 1:1000). Invitrogen: PTEN (44-1064; 1:1000). BD Biosciences: CTNNB1 (610153; 1:1000). Genetex: VPS35 (GTC108058; 1:1000).
Ctnnb1
Manufactured by Merck Group
Sourced in United States
CTNNB1 is a laboratory equipment product used for the detection and analysis of the beta-catenin protein. It is a key component of the Wnt signaling pathway, which plays a crucial role in various cellular processes. The core function of CTNNB1 is to provide researchers with a tool to study the expression and localization of beta-catenin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axin1, by Cell Signaling Technology (1 mentions)
Ctnnb1, by BD (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Criterion tgx precast gel, by Bio-Rad (1 mentions)
Ppm1a, by Abcam (1 mentions)
Vps35, by GeneTex (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab217668, by Abcam (1 mentions)
Ab137872, by Abcam (1 mentions)
Ab15251, by Abcam (1 mentions)
C2206, by Merck Group (1 mentions)
Epidermal growth factor (egf), by BD (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Ultra low attachment 6 well dishes, by Corning (1 mentions)
Dmem f12 medium, by BD (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Ab32072, by Abcam (1 mentions)
Phase contrast microscope, by Nikon (1 mentions)
Scrambled sirna, by Merck Group (1 mentions)
5 protocols using ctnnb1
1
Western Blot Analysis of Diverse Cellular Proteins
2
Transcriptional Regulation and Self-Renewal Assay
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Total mRNA was reverse‐transcribed into cDNA (AT301 TransGen Biotech, Beijing, China), and real‐time quantitative PCR was performed with CFX96 Real‐Time PCR Detection System (Bio‐Rid, Hercules, CA, USA). For western blot, the protein from cell extracts was separated by 10% SDS‐PAGE electrophoresis and was later transferred onto PVDF membrane. Membranes were incubated with ESR1 (1:3000, EPR4097, ab108398; Santa Cruz, Dallas, TX, USA), TCF4 (1:2000, ab217668; Abcam, Cambridge, MA, USA), CMYC (1:2000, ab32072; Abcam), LEF1 (1:2000, ab137872; Abcam), WNT1 (1:1000, ab15251; Abcam), CTNNB1 (1:1000, C2206; Sigma‐Aldrich, St. Louis, MO, USA), VINCULIN (1:5000, #18799; Cell Signaling Technology, Danvers, MA, USA), and then detected using ECL Blotting Detection Reagents (Merck Millipore, Burlington, MA, USA). Cells of different groups were suspended in DMEM/F12 Medium supplemented with 20 ng/mL EGF (BD Biosciences, San Jose, CA, USA), bFGF and 4 μg/mL insulin (Sigma), and then plated in 6‐well ultra‐low attachment dishes (1000 cells/mL; Corning Incorporated, Tewksbury, MA, USA). To analyse the self‐renewal ability, sphere number of each captured image was counted by using phase contrast microscope (Nikon, Tokyo, Japan).
3
Synthesis of UBE3C-LRP5 siRNAs
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Sense and anti-sense DNA oligonucleotides for UBE3C-LRP5 fusion variants (v1, v2, and v7), LRP5, CTNNB1, LEF1, and scrambled siRNA (Supplementary Table S5 ) were ordered from Sigma-Aldrich. The protocol for the synthesis of small RNA transcripts using T7 RNA polymerase is reported in the literature69 (link). For each in vitro transcription (IVT) reaction, 1 nmol of each oligonucleotide (re-suspended in 1X TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA)) was annealed using thermocycler to obtain double-stranded DNA (dsDNA). The thermocycler conditions used were: 95 °C for 3 min, followed by 70 cycles of 95 °C for 30 s (−1 °C/cycle). In vitro transcription (IVT) reaction was performed in 20 µL of a reaction containing 1X T7 transcription buffer (cat. no. P118B, Promega), 1X biotin RNA labeling mix (cat. no. 11685597910, Sigma-Aldrich), 1 U RiboLock RNase Inhibitor (cat. no. EO0381, Fermentas), 10 U T7 RNA polymerase (cat. no. P2075, Promega), and 1 nmol of dsDNA, as a template. The reaction was incubated at 37 °C for 2 h. Sense and anti-sense small interfering RNA (siRNA) synthesized in separate reactions were annealed by mixing the transcription reactions at 95 °C for 1 min, followed by 70 cycles of 95 °C for 30 s (−1 °C/cycle) to obtain double-stranded siRNAs.
4
Lentiviral shRNA Targeting β-catenin
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The shRNA clone for β-catenin (CTNNB1) was obtained from Sigma-Aldrich in the pLKO.1-puro vector (MISSION shRNA). The clone IDs were TRCN0000314991 and TRCN0000314921 for shCTNNB1-1 and shCTNNB1-2, respectively. The shRNA clones for protein phosphatase catalytic subunit a (PPP1Ca) and scramble control have been previously reported (Khaliq et al., 2020 (link)). Lentivirus packaging and testing were performed as described previously (Baek et al., 2018 (link)).
5
Lentiviral Transduction and Stable Cell Lines
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Lentiviral pLKO.1-puromycin negative control and human CDH2, CTNND1, CTNNB1, and CTNNA1 shRNA vectors were from Sigma-Aldrich (Table 2 ).
pLenti.CAG.H2B.Dendra2.W was from Addgene (RRID:Addgene_51005). N-cad WT or W161A mutated DNA was amplified by PCR from pCAG-Ncad WT or W161A-HA (Kon et al., 2019 (link)) and cloned into lentiviral pLenti-CAG-mCherry vector using NEBuilder HiFi DNA assembly (E2621; New England Biolabs) to generate pLenti-Ncad WT-HA-mCherry or pLenti-Ncad W161A-HA-mCherry.
To harvest lentiviral particles, lentiviral vector DNA was transfected with psPAX2 (RRID:Addgene_12260) and pMD2.G (RRID:Addgene_12259) packaging plasmids into HEK-293FT cells using Lipofectamine 2000 transfection reagent (11668019; Invitrogen) and D10 media lacking antibiotics. After 24 h, media were changed to glioma growth medium and the virus collected for a further 40 h. Culture media were collected and filtered through a 0.45-μM syringe filter. Glioma cells in a six-well plate were incubated with 500 μl of viral supernatant and 500 μl growth media for at least 48 h before selection. pLKO.1-shRNA-puromycin transduced cells were selected with 0.5 μM puromycin for 48 h. Cells expressing H2B-Dendra2, Ncad WT-HA-mCherry, or Ncad W161A-HA-mCherry constructs were sorted on a SONY MA900 Multi-Application Cell Sorter.
pLenti.CAG.H2B.Dendra2.W was from Addgene (RRID:Addgene_51005). N-cad WT or W161A mutated DNA was amplified by PCR from pCAG-Ncad WT or W161A-HA (Kon et al., 2019 (link)) and cloned into lentiviral pLenti-CAG-mCherry vector using NEBuilder HiFi DNA assembly (E2621; New England Biolabs) to generate pLenti-Ncad WT-HA-mCherry or pLenti-Ncad W161A-HA-mCherry.
To harvest lentiviral particles, lentiviral vector DNA was transfected with psPAX2 (RRID:Addgene_12260) and pMD2.G (RRID:Addgene_12259) packaging plasmids into HEK-293FT cells using Lipofectamine 2000 transfection reagent (11668019; Invitrogen) and D10 media lacking antibiotics. After 24 h, media were changed to glioma growth medium and the virus collected for a further 40 h. Culture media were collected and filtered through a 0.45-μM syringe filter. Glioma cells in a six-well plate were incubated with 500 μl of viral supernatant and 500 μl growth media for at least 48 h before selection. pLKO.1-shRNA-puromycin transduced cells were selected with 0.5 μM puromycin for 48 h. Cells expressing H2B-Dendra2, Ncad WT-HA-mCherry, or Ncad W161A-HA-mCherry constructs were sorted on a SONY MA900 Multi-Application Cell Sorter.
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