Total proteins from tissues were extracted using RIPA lysis buffer containing a mixture of protease inhibitors. Protein concentrations were measured using the BCA Protein Assay Kit according to the manufacturer’s protocol. After boiling the total protein, the proteins (20 μL per well) were separated in 10% SDS-PAGE and then transferred to PVDF membranes. To seal the proteins, the PVDF membranes were incubated with 5% fat-free milk for 90 min. Then, the membranes were incubated overnight at 4 °C using primary antibodies MUC16 (1:500, Abcam, UK), FOXA1 (1:1000, Abcam, UK), NDUFA (1:1000, Abcam, UK), COX15 (1:1000, Abcam, UK), β-actin (1:3000, Abcam, UK). After washing, the membrane was incubated with the appropriate secondary antibody for 2 h. After the detection of the signal using digital imaging equipment, the protein bands were quantified by ImageJ software.
Muc16
Manufactured by Abcam
Sourced in United Kingdom
MUC16 is a protein that is expressed on the surface of certain cells. It is a large, heavily glycosylated protein that is involved in cell-cell adhesion and signaling. MUC16 is often used as a biomarker for certain types of cancer.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Foxa1, by Abcam (1 mentions)
Hpv 16 e7, by Abcam (1 mentions)
Rabbit anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti mouse 115 035 003 and rabbit 111 035 003 secondary, by Jackson ImmunoResearch (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Immobilon p polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Akt 10176 2 ap, by Proteintech (1 mentions)
Pi3k bs 2067r, by Bioss Antibodies (1 mentions)
Gapdh d16h11, by Cell Signaling Technology (1 mentions)
P akt 66444 1 ig, by Proteintech (1 mentions)
Halt protease inhibitor and edta, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp, by Agilent Technologies (1 mentions)
Goat anti mouse hrp, by Agilent Technologies (1 mentions)
Ecl select western blotting detection reagent, by GE Healthcare (1 mentions)
Gapdh, by Merck Group (1 mentions)
6 protocols using muc16
1
Protein Extraction and Quantification
2
Antibody Sources and Cell Lines for HPVOPC Research
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The MUC16 (#X325) and HPV-16-E7 (#ab20191) antibodies were purchased from Abcam. The HLA-DRB1 antibody (#LS-C138934) was obtained from LS Biosciences. The Syntenin antibody (#H00006386) was purchased from Abnova and SIRPA antibody (#PA5-29544) from Thermoscientific. The HPV-16-L1 antibody (#NB120-3199) was obtained from Novus Biological. Anti-rabbit E-cadherin (#24E10) and Vimentin (#D21H3) antibodies were purchased from Cell Signaling Inc. Anti-mouse (#115-035-003) and rabbit (#111-035-003) secondary antibodies were obtained from Jackson Immunoresearch. The HMLE cells ware kindly provided by Dr. Guojun Wu, Wayne State University and the MCF-10A cells were obtained from ATCC. The HPV-16 integrated UM-SCC-104 cell line generated from a recurrent HPVOPC subject30 (link) was obtained from Dr. Thomas Carey, The University of Michigan Comprehensive Cancer Center. All cell lines were authenticated and periodically checked for mycoplasmal contamination using a mycoplasma detection kit (Sigma #MP-0025).
3
Western Blotting Analysis of Cell Signaling
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Western blotting analysis was performed using Immobilon-P polyvinylidene difluoride membranes (Millipore, 28820 Single Oak Drive, Temecula, California 92590, USA). The cell lysates were separated on sodium dodecyl sulfate–polyacrylamide gels (SDS PAGE) and western blotting analysis was performed with following antibodies against IGF2BP3 (Product #: 14,642–1-AP, 1:500 dilution) (Proteintech Group, Inc, USA), PI3K (bs-2067R, 1:1000 dilution) (Bioss, Beijing, China), p-PI3K (bs-5570R, 1:1000 dilution) (Bioss, Beijing, China), AKT (10,176–2-AP, 1:1000 dilution) (Proteintech Group, Inc, USA), p-AKT (66,444–1-Ig, 1:5000 dilution) (Proteintech Group, Inc, USA) or MUC16 (Ab205718, 1:500 dilution) (Abcam, Discovery Drive, Cambridge Biomedical Campus, Hills Road, Cambridge, CB20AX, United Kingdom, UK), respectively. Anti-alpha Tubulin antibody [DM1A]—Loading Control (Ab7291) at 1/1000 dilution (Abcam, Cambridge, United Kingdom, UK) and GAPDH (D16H11, 1:1000 dilution) (Cell Signaling Technology) was used as an internal control. The density of band was measured using the ChemiDoc™ XRS + System (1000 Alfred Nobel Drive, Hercules, California 94547, USA) equipping with Image-Pro Plus software and Epson color image scanner. The data were normalized to the Tubulin or GAPDH.
4
Western Blot Analysis of Epithelial Cell Proteins
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Protein was extracted from epithelial cells using RIPA buffer (Thermo Scientific) plus Halt Protease Inhibitor and EDTA (Thermo Scientific), and the concentration quantified using the Pierce BCA protein assay kit (Thermo Scientific). Western blotting was performed as previously described [20] using 40µg of each sample and the following primary antibodies: PAX6 (Covance, PRB-278P), GAPDH (Millipore, MAB374), MUC16 (Abcam, AB134093) and p63α (Cell Signaling Technology, 4892). Primary antibody dilutions used were: MUC16 (1:10,000); PAX6 (1:500); p63α
(1:800-1:100); and GAPDH (1:1500). The following secondary antibodies were used: goat antimouse HRP (P0447, Dako) (1:10,000) and goat anti-rabbit HRP (P0448, Dako) (1:5000-1:10,000).
Membranes were visualised using ECL Select western blotting detection reagent (GE Healthcare) and a Fuji image reader (LAS-1000 Pro version 2.1). Densitometric analysis was performed using Image J software, and results were normalised to GAPDH.
(1:800-1:100); and GAPDH (1:1500). The following secondary antibodies were used: goat antimouse HRP (P0447, Dako) (1:10,000) and goat anti-rabbit HRP (P0448, Dako) (1:5000-1:10,000).
Membranes were visualised using ECL Select western blotting detection reagent (GE Healthcare) and a Fuji image reader (LAS-1000 Pro version 2.1). Densitometric analysis was performed using Image J software, and results were normalised to GAPDH.
5
Protein Expression Profiling in Cells
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Equal amounts of proteins (20 μg) lysed from cells were resolved in SDS-PAGE gel and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). Primary antibodies against MUC16 (1:10000, Abcam, Cambridge, UK), p120-catenin (1:1000, Abcam, Cambridge, UK), FLAG (1:500, Proteintech Group, Inc., IL, USA), GAPDH (1:1000, Cell Signaling Technology, Beverly, MA), β-actin (1:1000, Abcam, Cambridge, UK), lamin B1 (1:500, Abcam, Cambridge, UK), Rac1 (1:1000, Abcam, Cambridge, UK), Cdc42 (1:125, Abcam, Cambridge, UK), and RhoA (1:5000, Abcam, Cambridge, UK) were used. The synthetic peptide of primary antibodies against MUC16 corresponding to Human MUC16 aa 12,450–12,550. Anti-rabbit or anti-mouse IgG (1:2000, Cell Signaling Technology, Beverly, MA) was used as the secondary antibody. For subcellular fractions, cytoplasmic or nuclear proteins were extracted using a cytoplasmic protein extraction kit or a nuclear protein extraction kit (SAB, MD, USA). The purity of subcellular fractions was confirmed using antibodies against GAPDH (a cytoplasmic marker) and lamin B1 (a nuclear marker).
6
Evaluate HCEC Differentiation and Stratification
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When the HCEC cells reached confluence on the transwell insert, the medium was removed to establish an ALI culture for promoting differentiation and stratification. After airlift culturing for two weeks to induce multi-layers of HCECs with or without hyperosmotic stress (400 mOsm/l) and diquafosol tetrasodium (100 µM) for 24 h, HCECs on the insert membrane were immunostained. Cells were fixed with cold methanol, permeabilized with 0.1% Triton X-100, and incubated with 10% goat serum for 1 h to block nonspecific reactions. Then, cells were incubated with MUC1 (Abcam), MUC5AC (Abcam), MUC16 (Abcam), and P2Y2R (Santacruz) antibodies and incubated with Alexa Fluor 488–conjugated anti-rabbit IgG antibody. The immunostains were captured using confocal microscopy (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).
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