The primary antibodies for PP2A-B55δ were purchased from Abcam (MA, USA) and Santa Cruz (CA, USA). PP2A-Aα subunit antibody was from Covance (NJ, USA). PP2A-C subunit, phosphorylated CDK1 (p-CDK1 Tyr15), CDK1, and cleaved Caspase-3 antibodies, and the secondary antibodies anti-rabbit IgG and anti-mouse IgG were from Cell Signaling Technology (MA, USA). PP2A-B55α antibody was from Merck Millipore (MA, USA). PP2A-B56γ antibody was obtained from Thermo. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and B-cell lymphoma 2-related protein (Bcl-2) antibodies were from Beyotime (Shanghai, China). Antibodies for Cyclin B1, Cyclin E1, Bcl-2-associated X protein (Bax), and proliferating cell nuclear antigen (PCNA) were from Ruiying Bioalogical (Jiangsu, China). The secondary antibodies anti-rat IgG and anti-goat IgG were from Proteintech (IL, USA). The fluorescein isothiocyanate-conjugated (FITC) rabbit anti-goat IgG was from Liankebio (Zhejiang, China).
Glyceraldehyde 3 phosphate dehydrogenase gapdh
Manufactured by Beyotime
Sourced in China
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme that catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate during the glycolysis process. It is a key enzyme in the metabolic pathway.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by R&D Systems (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Ire1α, by Abcam (1 mentions)
C jun, by Abcam (1 mentions)
P c jun, by Abcam (1 mentions)
Sqstm1 p62, by Abcam (1 mentions)
Anti mouse igg hrp, by Beyotime (1 mentions)
Lc3b 2, by Abcam (1 mentions)
P eif2α, by Abcam (1 mentions)
P jnk, by Abcam (1 mentions)
Beclin 1, by Abcam (1 mentions)
Grp78, by Abcam (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Magic mark xp western standard, by Thermo Fisher Scientific (1 mentions)
Cyclin e, by Abcam (1 mentions)
5 protocols using glyceraldehyde 3 phosphate dehydrogenase gapdh
1
Antibodies for PP2A Subunit Analysis
2
Western Blotting for Protein Expression
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Western blotting was used to detect expression levels of proteins as described previously
[18 (link),23 (link)]. We used antibodies against AQP3 (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin, E-cadherin, Snail, AKT, phospho-AKT(Ser473) (Cell Signaling Technology, Beverly, MA), fibronectin (R&D systems, Minneapolis, MN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime Institute of Biotechnology, Henan, China). Densitometric analysis of proteins was conducted and normalized against GAPDH. The PI3 kinase inhibitor LY294002, was obtained from Cell Signaling Technology (Beverly, MA).
[18 (link),23 (link)]. We used antibodies against AQP3 (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin, E-cadherin, Snail, AKT, phospho-AKT(Ser473) (Cell Signaling Technology, Beverly, MA), fibronectin (R&D systems, Minneapolis, MN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime Institute of Biotechnology, Henan, China). Densitometric analysis of proteins was conducted and normalized against GAPDH. The PI3 kinase inhibitor LY294002, was obtained from Cell Signaling Technology (Beverly, MA).
3
Apoptosis and Autophagy Pathway Analysis
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XAG (HLPC ≥98%, MW: 392.49) was synthesized as previously described [22 (link)]. Specific antibodies against cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved caspase-12, cleaved PARP, Bcl-2, Bak, Bax, LC3B-II, p62/SQSTM1, Beclin-1, Atg5, p-JNK, JNK, p-c-jun, c-jun, Ki-67, CHOP, GRP78, ATF6, p-eIF2α, IRE1α were purchased from Abcam (Cambridge, UK), specific antibody against cytochrome C was obtained from Cell Signaling (Danvers, MA, USA). Antibody against COX-IV was purchased from Abcam (Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and goat anti-rabbit immunoglobulin horse radish peroxide (IgG-HRP) or anti-mouse IgG-HRP were obtained from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Fluorescent antibody against LC3 was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China).
4
Siglec15 Protein Expression Analysis
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Tissue or cell proteins were collected using radio-immunoprecipitation assay (RIPA) lysis buffer, and a bicinchoninic acid (BCA) assay was applied to quantify the concentration of proteins. After boiling, the same amount of protein was separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk at room temperature and probed with primary antibodies for Siglec15 (Sigma Aldrich, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, China) overnight at 4 ℃, followed by incubation with secondary antibodies at 25 ℃ for 2 h. The signals were visualized using a chemiluminescence system with the enhanced chemiluminescence (ECL) reagent (WBKlS0100, Millipore, USA).
5
Western Blot Analysis of Cell Cycle Regulators
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Cells were lysed in a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) with complete protease inhibitor cocktail (Roche, Basel, Switzerland), and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using MagicMark XP Western Standard (Invitrogen). Then, membranes were incubated with a primary antibody against human E2F5 (1:1000; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000; Beyotime, Shanghai, China), CDK2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDK4 (1:500; Santa Cruz Biotechnology), Cyclin D1 (1:500; Santa Cruz Biotechnology), or Cyclin E (1:500; Abcam) at 4°C overnight. After three 10-min washes with tris-buffered saline with Tween 20 (TBST), the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature (1:1000; Santa Cruz Biotechnology). The targeted bands were visualized with enhanced chemiluminescence (ECL).
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