All tumor samples and control tissues were fixed overnight in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Hematoxylin and eosin (H&E)-stained sections were used for diagnostic purposes and unstained sections for immunohistochemical studies. Protein concentration was determined, and equal amounts of total proteins were separated on SDS- PAGE. IHC was performed with the following antibodies: rabbit polyclonal antibody for Cleaved Caspase-3 (Cell Signaling) and Ki-67 (Santa Cruz Biotechnology). The evaluation of the IHC was conducted blindly, without knowledge of the treatment. Immunoblot analyses were performed on lysates extracted from tumors. Antibodies used for western blot included PAK1, phospho-PAK1, Cleaved Caspase-3, H2AX and phospho-H2AX from Cell Signaling Technology; Rad51, FANCI, FANCD2 and Ki-67 were from Santa Cruz Biotechnology. GAPDH was used as loading control.
Fanci
Manufactured by Santa Cruz Biotechnology
Sourced in United States
FANCI is a laboratory instrument designed for the detection and analysis of proteins and nucleic acids. It utilizes advanced imaging and detection technologies to provide accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Fancd2, by Santa Cruz Biotechnology (2 mentions)
Phospho h2ax, by Cell Signaling Technology (1 mentions)
Phospho pak1, by Cell Signaling Technology (1 mentions)
Rad51, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Boster Bio (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Impdh2, by Proteintech (1 mentions)
Mek1 2, by Proteintech (1 mentions)
Phospho mek1 2, by Cell Signaling Technology (1 mentions)
Erk1 2, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Cyclin a, by GeneTex (1 mentions)
Vinculin, by Merck Group (1 mentions)
Palb2, by Fortis Life Sciences (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
Ph2ax, by Merck Group (1 mentions)
Ubiquityl pcna lys164, by Cell Signaling Technology (1 mentions)
4 protocols using fanci
1
Immunohistochemical and Immunoblot Analysis of Tumor Samples
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using RIPA buffer (Boster, Wuhan, China) containing the protease inhibitor PMSF (Boster). Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. The blots were blocked by incubation with 5% fat-free milk at room temperature for 2 h and then incubated overnight at 4°C with a 1:500 dilution of antibodies to the following proteins: FANCI (Santa Cruz Biotechnology, Dallas, TX, USA), IMPDH2, MEK1/2, ERK1/2, MMP2, MMP9, GAPDH (all Proteintech, Wuhan, China), phospho (p)-MEK1/2, and p-ERK1/2 (both Cell Signaling Technology, Danvers, MA, USA). The membranes were washed three times with TBST and then incubated for 2 h with horseradish peroxidase-conjugated rabbit or mouse secondary antibodies. After signal development, expression of proteins was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Analysis of DNA Repair Proteins
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Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used. Secondary antibodies were conjugated with horse radish peroxidase (mouse or rabbit, Cell Signaling Technology) or appropriate Alexa Fluor (Molecular Probes) were used.
4
Western Blot Analysis of DNA Repair Proteins
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Whole-cell lysates were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris [pH 7.4], 150 mM sodium chloride [NaCl], 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Protein from the insoluble fraction was extracted from the cell pellet using a solubilization solution (8 M urea, 10% 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride [PMSF]) and incubated at 37°C for 30 min. Protein was quantitated using a Bradford assay (Bio-Rad) and run on a Tris-glycine sodium dodecyl sulfate (SDS)-polyacrylamide gel. Protein was transferred to an Immobilon-P polyvinylidene fluoride membrane (Millipore) and probed with primary and secondary antibodies. ECL (enhanced chemiluminescence) or ECL Prime (GE) was used to visualize protein. The antibodies used were FANCD2 (Abcam no. ab108928; Thermo Scientific catalog no. MA1-16570), cytokeratin 10 (Santa Cruz catalog no. sc52318), γH2AX-Ser139 (Cell Signaling catalog no. 5438), FANCI (Santa Cruz catalog no. sc-98532), BRCA1 (Cell Signaling catalog no. 9010), RAD51 (Cell Signaling catalog no. 8875), BRCA2 (Cell Signaling catalog no. 9012), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz catalog no. sc-47724).
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