The human Cervical Cancer cells were lysed with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 48 h after transfection. The protein concentration was measured using Lowry's method. Total proteins (50 μg per lane) were separated by using 12% SDS-polyacrylamide gel electrophoresis and then were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated at room temperature for 1 h with 5% skimmed milk powder to block non-specific antibody binding. Primary rabbit monoclonal antibody against human AEG-1 (ab124789) (1:10,000; Abcam, Cambridge, MA, USA) or mouse monoclonal antibody against human β - actin (ab6276) (1:5,000; Abcam, Cambridge, MA, USA) and mouse monoclonal antibodies HPV 16-E6 (sc-460), HPV 16-E7 (sc-65711), HPV 18-E6 (sc-365089), HPV 18-E7 (sc-365035) (1:500; Santa Cruz Biotechnology, St Louis, CA) were incubated overnight at 4°C and then incubated for 1 h at room temperature with a goat anti-rabbit (ab6722) or anti-mouse (ab97020) alkaline phosphatase tagged secondary antibody (1:1,000; Abcam, Cambridge, MA, USA). Bands detected using the of BCIP/NBT solution (Merck Millipore, Bedford, MD, USA) were visualized using Bio-Rad Gel doc XR plus (Bio-Rad, Hercules, CA, USA) and band intensities were quantified using ImageJ software (NIH).
Ab6722
Manufactured by Abcam
Sourced in United States
Ab6722 is a monoclonal antibody that recognizes the BRCA1 protein. It is suitable for use in various applications, including Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Ab124789, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ac026, by ABclonal (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Sc 365513, by Santa Cruz Biotechnology (1 mentions)
Bs0162r, by Bioss Antibodies (1 mentions)
Bs 6309r, by Bioss Antibodies (1 mentions)
Bsm 33207m, by Bioss Antibodies (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Donkey anti goat igg h l alexa fluor 488, by Abcam (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
D9542, by Merck Group (1 mentions)
Oct compound, by Leica (1 mentions)
Ab150129, by Abcam (1 mentions)
Ab66047, by Abcam (1 mentions)
7 protocols using ab6722
1
Western Blot Analysis of Cervical Cancer Cells
2
Protein Isolation and Western Blot Analysis
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Total protein samples were isolated from tissue samples using RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) containing both PMSF and phosphatase inhibitors for western blot analyses. A BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Rockford, IL, United States) was used to determine the protein concentration. The protein samples were separated on the 10% or 12% SDS-PAGE gels, and then transferred to a polyvinylidene fluoride (PVDF, Millipore, MA, United States) membranes. After blocking with 5% skim milk, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies: anti-TNFR1 (1:1000, 21574-1-AP, Proteintech, China), anti-phospho-NF-κB p65(Ser536) (1:1000, 8324, CST), anti-IL1β (1:1000, ab6722, Abcam, United States), IL6 (1:1000, bs-6309R, Bioss), iNOS (1:1000, bs-0162R, Bioss), anti-TNFα (1:1000, bsm-33207M, Bioss), anti-SREBP-1 (1:1000, sc-365513, Santa Cruz Biotechnology), anti-PPARγ (1:1000, bsm-4590R, Bioss) and β-actin (1:5000, AC026, ABclonal, China). After that, the PVDF membranes were incubated with the corresponding secondary antibodies. An ECL kit (Tanon, Shanghai, China) was used to visualize the chemiluminescence signals. Protein expression levels were quantified with ImageJ software. The housekeeping protein β-actin was utilized as loading control.
3
Immunohistological Analysis of Brain Sections
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For cryoprotection, brains were gradually immersed in 10, 20, and 30% sucrose in 24 h intervals and embedded in OCT compound (Leica Microsystems, Bensheim, Germany) with liquid nitrogen. The brains were cut into frozen coronal sections (30 μm) using a Leica CM3050 cryostat and then stored in a free-floating buffer. For immunohistological analysis, the sections were blocked in 5% normal chicken serum (containing 0.3% Triton X-100) for 1 h. After washing, the sections were incubated with primary antibodies against ionized calcium-binding adaptor molecule 1 (Iba-1, 1:200, 019-19741, Wako) or 5-hydroxytryptamine (5-HT, 1:200, ab66047, Abcam) overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG HRP (1:400, ab6722, Abcam) or Alexa Fluor® 488 donkey anti-goat IgG H&L (1:400, ab150129, Abcam) secondary antibodies for 2 h at RT. To analyze the Iba-1-positive cells, the sections were incubated with an avidin-biotin-peroxidase complex (VECTASTAIN ABC kit, Vector Laboratories) for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. To analyze the 5-HT-positive signal, the sections were exposed to DAPI (1:1,000, D9542, Sigma) to stain cell nuclei. Immunoreactivity was observed under an Axio-Phot microscope (Carl Zeiss, Germany). The intensity was quantified using ImageJ 1.46 software (NIH, Bethesda, MD, USA).
4
Quantifying Liver Protein Expression
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To evaluate the presence of alpha smooth muscle actin (α-SMA) and TGF-β in the liver parenchyma, 100 μg of tissue was homogenized in 1 mL of lysis buffer (50 mM Tris-HCl at pH 6.8, 5 mM N-ethylmaleimide, 3 mM iodoacetamide, 1 mM phenylmethanesulfonyl fluoride, and 3 mM tosyl-L-lysine chloromethyl ketone) for total protein extraction. The lysate was centrifuged at 40,000 g for 1 h at 4°C. The supernatants were suspended with 200 μL lysis buffer and 1% triton X-100. Protein quantification was performed by the Bradford method.
For Western blot, 50 μg of each protein extract was separated in a 12% SDS-PAGE gel and proteins were transferred to a PVDF membrane (Bio-Rad, 162-0176, Hercules, USA). Blockage was carried out for 1 h at room temperature with TBST (Tris-buffered saline/0.05% Tween-20) and 5% skimmed milk. For immunodetection, the membrane was incubated for 1 h at room temperature with the following antibodies diluted at 1:1000: mouse anti-human TGF-β (Peprotech H2614), rabbit polyclonal anti-α-SMA (Abcam ab5694), and rabbit polyclonal anti β-actin (Abcam ab69512). Then, blots were incubated with the conjugated antibody, goat anti-mouse, and goat anti-rabbit marked with alkaline phosphatase 1:2000 (Sigma A3688, Abcam ab6722). After three washes with TBS, blotting was developed by alkaline phosphatase with the sigma fast bcip/nbt (Sigma).
For Western blot, 50 μg of each protein extract was separated in a 12% SDS-PAGE gel and proteins were transferred to a PVDF membrane (Bio-Rad, 162-0176, Hercules, USA). Blockage was carried out for 1 h at room temperature with TBST (Tris-buffered saline/0.05% Tween-20) and 5% skimmed milk. For immunodetection, the membrane was incubated for 1 h at room temperature with the following antibodies diluted at 1:1000: mouse anti-human TGF-β (Peprotech H2614), rabbit polyclonal anti-α-SMA (Abcam ab5694), and rabbit polyclonal anti β-actin (Abcam ab69512). Then, blots were incubated with the conjugated antibody, goat anti-mouse, and goat anti-rabbit marked with alkaline phosphatase 1:2000 (Sigma A3688, Abcam ab6722). After three washes with TBS, blotting was developed by alkaline phosphatase with the sigma fast bcip/nbt (Sigma).
5
Quantifying HTLV-1 Antibody Response in Rabbits
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The HTLV antibody response was assessed qualitatively in a representative rabbit from each condition via a modified MP Diagnostics HTLV Blot 2.4 Western Blot Assay protocol (MP Biomedicals LLC, Santa Ana, CA). The supplied alkaline phosphatase conjugated goat anti-human immunoglobulin gamma (IgG) was substituted for an alkaline phosphatase conjugated goat anti-rabbit IgG (ab6722; Abcam, Cambridge, United Kingdom). Plasma from each condition was diluted 1:10.
After qualitative assessment of representative rabbits, HTLV-1-specific antibody response was quantified for all rabbits using a modified Avioq HTLV-1/2 Microelisa System protocol (Avioq, Inc., Research Triangle Park, NC). The supplied horseradish peroxidase (HRP) conjugated goat anti-human IgG was substituted for an HRP conjugated goat anti-rabbit IgG (ab6721; Abcam, Cambridge, United Kingdom). Rabbit plasma was diluted 1:500 to obtain absorbance values within the linear range of the assay.
Total rabbit IgG was quantified using the Abcam Rabbit IgG ELISA Kit in accordance with the provided protocol (ab187400; Abcam, Cambridge, United Kingdom). Plasma samples were diluted 1:1 × 106. Mixed model analyses with a Bonferroni correction were performed in weeks 8 and 12 (HTLV-1-specific) or 2 and 12 (total rabbit IgG) to determine statistical significance. A p < 0.0083 was considered a statistically significant change.
After qualitative assessment of representative rabbits, HTLV-1-specific antibody response was quantified for all rabbits using a modified Avioq HTLV-1/2 Microelisa System protocol (Avioq, Inc., Research Triangle Park, NC). The supplied horseradish peroxidase (HRP) conjugated goat anti-human IgG was substituted for an HRP conjugated goat anti-rabbit IgG (ab6721; Abcam, Cambridge, United Kingdom). Rabbit plasma was diluted 1:500 to obtain absorbance values within the linear range of the assay.
Total rabbit IgG was quantified using the Abcam Rabbit IgG ELISA Kit in accordance with the provided protocol (ab187400; Abcam, Cambridge, United Kingdom). Plasma samples were diluted 1:1 × 106. Mixed model analyses with a Bonferroni correction were performed in weeks 8 and 12 (HTLV-1-specific) or 2 and 12 (total rabbit IgG) to determine statistical significance. A p < 0.0083 was considered a statistically significant change.
6
Plasma Membrane Protein Isolation and Analysis
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Plasma membrane enriched fraction was isolated following protocol described by Santoni (2007) (link) and protein was isolated in extraction buffer (50 mM NaHPO4, pH 7.0, 10 mM b-mercaptoethanol, 10 mM Na2EDTA, pH 8.0, 0.1% Sarcosyl, 0.1% Triton X-100). Total Proteins from wt Col-0, OE6.4, and OE11.4, as well as their plasma membrane enriched fraction (20 μg), were resolved on 12% (w/v) SDS-PAGE, transferred on to PVDF membrane and blocked with 4% (w/v) BSA at RT for 1 h. The membrane was labeled with an antibody against PIP2A (Agrisera- AS09491) or GFP (Abcam-183734) for overnight at 4°C. After washing in TBST-buffer (Tris-buffered saline, 0.1% Tween 20), the membrane was incubated in rabbit anti-goat HRP conjugate antibody (Abcam- ab6722) in TBS for 2–3 h at RT and washed again with TBST-buffer. Signal was detected using ECL solution.
7
Western Blot for Tetanus Neurotoxin
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Immunotransfer was undertaken using an iblot system (Invitrogen) with nitrocellulose membranes according to manufacturer's protocol. The nitrocellulose membrane was blocked with blocking buffer (5% Skim milk powder in Tris-buffered saline (TBS)) for 1 h. All incubations and washes were performed on a rotating shaker. The membrane was washed twice in TBS for 2 min, and then the primary antibody (Rabbit anti-TeNT IgG, ab53829, Abcam) diluted to 1:10,000 in blocking buffer was added for 2 h at room temperature (or overnight at 4oC). The blot was washed twice in TBS for 5 min, and the secondary antibody (Goat anti-rabbit IgG conjugated with AP, ab6722, Abcam) diluted to 1:10,000 in blocking buffer was added and incubated at room temperature for up to 2 h. The blot was washed three times in TBS 0.05% tween for 5 min. To develop the AP conjugate, the blot was immersed in Western Blue TM (Promega) solution for 5-10 min. until the desired resolution was achieved. The blot was washed in dH2O to stop the reaction.
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