Ribociclib (S7440) and Infigratinib (S2183) were purchased from Selleck Chemicals and were dissolved in 21% Captisol and 30% PEG300 solution (vehicle) for oral administration. Antibodies against FGFR1 (#9740), FGFR3 (#4574), FGFR4 (#8562), AKT (#9272), Rb (#9313), cyclin B1 (#4138), Cdc25C (#4688), Cyclin D1 (#2978), Cyclin E2 (#4132), survivin (#2803), Sox9 (#82630), cleaved PARP (#5625), β catenin (#8480), α‐tubulin (#2144) and phosphorylation‐specific antibodies against AKT Ser473 (#9271), FRS2‐α Tyr439 (#3861), Rb Ser807/811 (#9308), Histone 3 Ser10 (#9701), Cdc2 Tyr15 (#9111) and Erk1/2 Thr202/Tyr204 (#4370) were obtained from Cell Signaling Technology. Antibodies against CYP3A4 (ab124921) and HNF4‐α (ab201460) were from Abcam. The antibodies against FGFR2 (#sc‐122), Erk1/2 (#sc‐94), FRS2‐α (#sc‐17841), GAPDH (sc‐166545) and Cdk2 Thr14/Tyr15 (#sc‐28435‐R) were from Santa Cruz Biotechnology Inc. Anti‐mouse CD31 antibody (#2502) was from BioLegend. Anti‐albumin (#SAB4200711; clone HAS‐11) antibody was purchased from Sigma‐Aldrich.
Gapdh sc 166545
Manufactured by Santa Cruz Biotechnology
Sourced in United States
GAPDH (sc-166545) is a purified antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in the glycolytic pathway, catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
Ab201460, by Abcam (1 mentions)
Ab124921, by Abcam (1 mentions)
Frs2 α tyr439, by Cell Signaling Technology (1 mentions)
Histone 3 ser10, by Cell Signaling Technology (1 mentions)
Cdc2 tyr15, by Cell Signaling Technology (1 mentions)
Anti mouse cd31 antibody, by BioLegend (1 mentions)
Infigratinib, by Selleck Chemicals (1 mentions)
Ribociclib, by Selleck Chemicals (1 mentions)
Zb 2305, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Las4000 digital image scanning system, by GE Healthcare (1 mentions)
Parp 1 9542, by Cell Signaling Technology (1 mentions)
Sc 32245, by Santa Cruz Biotechnology (1 mentions)
5 protocols using gapdh sc 166545
1
Examining Targeted Cancer Therapy Mechanisms
2
Immunoblotting Technique with GAPDH, p53, and CDK1
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Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc166545, 1:3000) and the tumor protein, p53 (DO-1; sc-126, 1:1000), were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against cyclin-dependent kinase 1 (CDK1) (ab18, 1:1000 dilution) were purchased from Abcam (Cambridge, UK). Secondary horseradish peroxidase-conjugated antibodies against mouse immunoglobulin (Ig) G (ZB2305, 1:5000) were purchased from Zhongshan Golden Bridge Biotechnology. Co., LTD (Guangzhou, China).
3
Quantitative Western Blot Analysis
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Protein concentrations were determined with the BCA assay (Thermo Scientific, Waltham, MA, USA). Each lysate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and probed with rabbit monoclonal antibodies against RUNX2 (Cell Signaling, 8486), mouse monoclonal antibodies against RUNX2 (D130-3, clone 8G5; MBL), N-Cadherin (EPR1791-4), anti E-Cadherin antibody (EP700-Y Abcam, Cambridge, UK), anti-Vimentin antibody (EPR3776 Abcam) anti p53 (pAb122 Abcam), and GAPDH SC-166545 (Santa Cruz Biotechnology, Dallas, TX, USA) according to manufacturer’s instructions. Immuno-reactive proteins were detected using an enhanced chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. Images were captured either on film or by LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed by using ImageJ software (v 1.43o8, NIH, Bethesda, MD, USA), and the relative protein band intensity was normalized to GAPDH and expressed as OD ratio. Data were obtained from three independent experiments.
4
Measurement of Oxidative Stress Markers in Cells
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AQP4 (sc-20812), cPLA2 IVA (sc-454), ser 505 p-cPLA2 IVA (sc-34391), and glyceraldehyde phosphate dehydrogenase (GAPDH, sc-166545) were from Santa Cruz Biotechnology, Santa Cruz CA; iPLA2 VIβ (07-169) was from Upstate Biotech, Lake Placid NY; sPLA2 IIA (181055100) was from BioVendor, Candler NC; PARP-1 (9542s) was from Cell Signaling, Danvers MA; Anti-3NT-protein (AB7048) was from Abcam Co., Cambridge MA; and anti-4HNE-adducted protein (AB5605) was from Millipore Corp., Temecula CA. Secondary antibodies were from Jackson ImmunoResearch, West Grove PA, and luminol reagent for immunoblot detection was from Pierce Chemicals, Rockford IL. DHA was from Cayman Chemicals, Ann Arbor MI, PJ34 was from Enzo Life Sciences, Farmingdale NY, and other chemicals/reagents were from Sigma Chemicals, St. Louis MO.
5
Establishing LKB1-Deficient A549 Cell Line
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The adenocarcinoma cell line A549 was purchased from the American Type Culture Collection. Cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium purchased from Hyclone and supplemented with 10% fetal bovine serum; cells were grown at 37 °C in a humidified atmosphere with 5% CO2. Mouse monoclonal antibodies against LKB1 (sc-32245) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-166545) were purchased from Santa Cruz Biotechnology. A549 cells, a cell line with endogenous LKB1 deficiency, were transfected with pLenti-EF1a-mcherry-P2A-Puro-CMV-MCS-3Flag (control) or pLenti-EF1a-mcherry-P2A-Puro-CMV-stk11 stable plasmids. The cells were then subjected to puromycin selection (4 ng/μl) for 2 weeks, after which we collected puromycin-resistant stable clones. The expression of LKB1 in established stable transfected A549 cells was validated by Western blot. We also transfected the LKB1 (K78I) kinase-dead mutant plasmid into the A549 cell line by means of transient transfection and used the A549 cell line transfected with PCDNA3.0 vector plasmid as a control. We verified the expression of LKB1 by western blot.
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