For Western blotting, total protein lysate was extracted by RIPA buffer, and the nuclear/cytoplasmic protein fractions were extracted by NE-PER™ nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s instructions. Protein samples were separated by SDS-PAGE, transferred to polyvinyl difluoride membranes, blocked with 5% non-fat milk, and incubated with primary antibodies against the following antigens at 4°C overnight: β-catenin (1:1000, CST, USA), SNAIL (1:1000, CST), phospho-β-catenin-Tyr654 (1:1000, Abcam, USA), phospho-ERK1/2 (1:1000; Abcam), total-ERK (1:1000; Abcam), histone H3 (1:1000; Abcam), phospho-GSK-3β (1:1000, Abcam), β-Actin (1:1000, Abcam), and GAPDH (1:2500, Abcam). The next day, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500; Abcam). Electrochemiluminescence was assessed to visualize the protein bands (Abcam). GAPDH expression was used as an internal standard for total protein lysate. histone H3 and β-Actin expression levels were used as internal standards for nuclear proteins and cytoplasmic proteins, respectively.
Phospho gsk 3β
Manufactured by Abcam
Sourced in United States
Phospho-GSK-3β is a primary antibody that recognizes the phosphorylated form of glycogen synthase kinase-3 beta (GSK-3β). GSK-3β is a serine/threonine protein kinase involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Phospho erk1 2, by Abcam (1 mentions)
Total erk, by Abcam (1 mentions)
Ne per, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Histone h3, by Abcam (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Traf6, by Santa Cruz Biotechnology (1 mentions)
Traf3, by Santa Cruz Biotechnology (1 mentions)
Antibodies against β catenin, by Abcam (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Nfatc1, by Santa Cruz Biotechnology (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
4 protocols using phospho gsk 3β
1
Protein Expression and Fractionation
2
Molecular Mechanisms of Osteoclast Differentiation
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Cyclophosphamide (CPD) was purchased from Shengdi Company (Jiangsu, China). M-CSF and RANKL were purchased from Peprotech (New Jersey, USA). Primary antibodies against active β-catenin, Runx2, Cyclin D1, c-Myc, IκBα, phospho-IκBα, JNK, phospho-JNK, p38 and phospho-p38 were purchased from Cell Signaling Technology (Beverly, MA, USA) and others against TRAF-6, TRAF-3, NFATc1, RANK and c-Fos were purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-catenin, phospho-Gsk-3β, and DKK1 were purchased from Abcam Company (Cambridge, UK).
3
Autophagy Regulation Signaling Pathway
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3‐methylamphetamine (M9281), rapamycin (37094), SB216763 (S3442), and KT‐5823 (K1388) were purchased from Sigma‐Aldrich (St. Louis, MO, USA); antibodies were used against light‐chain 3B (LC3B; Aviva Systems Biology Cat# ARP51335_P050, https://scicrunch.org/resources/Any/search?q=AB_2044956&l=AB_2044956 ), Beclin 1 (Novus Cat# NB500‐249B, https://scicrunch.org/resources/Any/search?q=AB_1216268&l=AB_1216268 ), GSK‐3β (Cell Signaling Technology Cat# 12456, https://scicrunch.org/resources/Any/search?q=AB_2636978&l=AB_2636978 ), phospho‐GSK‐3β (Ser9; Cell Signaling Technology Cat# 14310, https://scicrunch.org/resources/Any/search?q=AB_2798445&l=AB_2798445 ), phospho‐GSK‐3β (Tyr216; Abcam, Cat# 75745), SQSTM1/p62 (Cell Signaling Technology Cat# 5114, RRID:AB_10624872), APG5L/ATG5 (Abcam Cat# ab109490, https://scicrunch.org/resources/Antibodies/search?q=AB_10861245&l=AB_10861245 ), https://scicrunch.org/resources/Antibodies/search?q=AB_1063421&l=AB_1063421 (LifeSpan Cat# LS‐C42463‐50, RRID:AB_1063421), https://scicrunch.org/resources/Antibodies/search?q=AB_10800793&l=AB_10800793 (LifeSpan Cat# LS‐C121392‐50, RRID:AB_10800793), https://scicrunch.org/resources/Antibodies/search?q=AB_2263076&l=AB_2263076 (Proteintech Cat# 10494‐1‐AP, RRID:AB_2263076), and β‐actin (Proteintech, Cat# 60008‐1‐Ig).
4
Immunohistochemical Analysis of Oncogenic Pathways
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Tumour tissues were deparaffinized, dehydrated, incubated with 3% H2O2, and treated with heated ethylene diamine tetraacetic acid for antigen retrieval. Then, a horseradish peroxidase/diaminobenzidine detection IHC kit was used according to the manufacturer’s instructions (Abcam). The antibodies used in this study were against the following antigens: β-catenin (1:200; Abcam), phospho-ERK1/2 (1:100; CST), phospho-GSK-3β (1:100, Abcam), and SNAIL (1:100; Abcam).
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