Total protein was extracted for western blotting analysis. The PVDF membranes were incubated overnight at 4°C with primary antibodies against COL6A6 (1:1000, PA5-60958, Thermo, USA), P4HA3 (1:1000, ab101657, Abcam, Cambridge, UK), E-cadherin (1:1000, ab1416, Abcam, Cambridge, UK), N-cadherin (1:1000, ab202030, Abcam, Cambridge, UK), Vimentin (1:1000, ab193555, Abcam, Cambridge, UK), p-PI3K(p85)(1:1000, #ab191606, Abcam, Cambridge, UK), PI3K(p85) (1:1000, #ab191606, Abcam, Cambridge, UK), and p-Akt (), Akt (), and subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (1:500, Abcam, Cambridge, UK). Signals were visualized using enhanced chemiluminescence reagent (Bio-Rad, USA).
Ab191606
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab191606 is a recombinant monoclonal antibody that specifically recognizes the protein target. The antibody is produced in a mammalian cell line and is purified by affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab182651, by Abcam (33 mentions)
Ab179463, by Abcam (18 mentions)
Ab8805, by Abcam (17 mentions)
Ab38449, by Abcam (15 mentions)
Ab192623, by Abcam (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Ab8245, by Abcam (7 mentions)
Ab81283, by Abcam (7 mentions)
Ab32503, by Abcam (7 mentions)
Ab32028, by Abcam (6 mentions)
Ab109268, by Abcam (6 mentions)
Ab181602, by Abcam (5 mentions)
Ab205718, by Abcam (5 mentions)
Ab9485, by Abcam (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
Ab134903, by Abcam (4 mentions)
Ab7817, by Abcam (4 mentions)
Ab8226, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab2302, by Abcam (4 mentions)
70 protocols using ab191606
1
Protein Expression Analysis by Western Blot
2
Evaluating Immune Signaling Molecules in HLH
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Peripheral blood samples were collected from the proband and parents, as well as a familial HLH patient and a healthy child as control. Peripheral blood mononuclear cells were isolated using lymphocyte separation solution. Protein expression in peripheral blood mononuclear cells was determined via flow cytometry. Briefly, cells were fixed and permeabilized sequentially with an IntraPrep Permeabilizaton Reagent kit (Beckman Coulter，A07803) following manufacturer’s instruction. Specific antibodies were then added for incubation overnight, followed by 30 mins incubation with a fluorescently labeled secondary antibody. The flow cytometry tests were carried out with a BD FACSCanto II flow cytometer, and the data was processed with CytExpert 2.0 (Beckman Coulter) software. Antibodies used were listed as follows: Anti-SH2D1A/SAP (Abcam, ab109120); Anti-PI3 Kinase p110δ (Abcam, ab109006); Anti-PI3 Kinase p85α (Abcam, ab191606); Anti-PTEN (Abcam, ab32199); Anti-AKT (CST, 9272s); Anti-phospho-AKT (CST, 9271s); Anti-mTOR (Abcam, ab2732).
3
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total protein was extracted using the radio-immunoprecipitation assay cell lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China) containing total protein. After concentration determination, an equal volume of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked by 5% non-fat milk and co-cultured with the primary antibodies against E-cadherin (1:1000, #3195, Cell Signaling Technology (CST), Beverly, MA, USA), Vimentin (1:1000, #5741, CST), Snail (1:1000, #3879, CST), GAPDH (1:1000, #5174, CST), p-protein kinase B (p-AKT1, phospho S473) (1:1000, ab81283, Abcam Inc., Cambridge, MA, USA), p-phosphatidyl inositol 3-kinase (p-PI3Kinase p85 alpha, 1:1000, ab191606, Abcam) and IGF2BP2 (1:1000, ab129701, Abcam) at 4 °C overnight, and then with secondary antibody IgG H&L (HRP) (1:10000, ab205718, Abcam) at 20 °C for 2 h. The protein blots were visualized using the Immobilon ECL substrate (Millipore), and the images were captured using an Optimax X film processor. Relative protein expression was examined using Image J (NIH), and GAPDH was used for internal loading.
4
Protein Expression Analysis of Rat Endometrium
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Rat endometrium tissues were initially homogenized and lysed in radio-immunoprecipitation assay (RIPA) buffer (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China) supplemented with proteinase inhibitor (Lot#4693116001, Roche). Lysates were then centrifuged for 20 min at 12,000 g. The bicinchoninic acid (BCA) (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China) was used for quantification of protein concentration. Equivalent amounts of protein (20 μg) were then used for western blot with primary antibodies of VEGF (ab53465, Abcam, UK), PI3K (ab191606, Abcam, UK), P-PI3K (ab182651, Abcam, UK), AKT (ab8805, Abcam, UK), P-AKT (ab38449, Abcam, UK), Ang-1 (ab102015, Abcam, UK), Ang-2 (ab155106, Abcam, UK) and HIF-1a (ab1, Abcam, UK) at 4 °C with gentle shaking overnight. After washing with TBS-T for three times, the membranes were then incubated with the secondary antibody at RT for 1 h and detected using an ECL plus kit (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China). The ECL signals were detected with Quantity One software (Bio-Rad, Hercules, CA) and blot intensities were quantified by using imageJ (NIH, Bethesda, MD) software. Tubulin (ab8245, Abcam, UK) was used as an internal control to validate the amount of protein loaded onto the gels.
5
Western Blotting Analysis of Hippocampal Proteins
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After the behavioral experiment, three mice were randomly selected from each group for Western blotting. The brain tissue of the mice was quickly removed and the hippocampus tissue was separated at low temperature. The treated brain tissue requires extraction of histones at low temperature, followed by grinding and centrifugation of the sample. The protein concentration of the sample is determined from the standard curve and then heated to denature the proteins of the sample. Electrophoresis and membrane transfer are then performed. We use the first antibody to react with the PVDF membrane overnight, put down the second antibody the next day and finally rinse with PBS solution. The gels were then scanned using an automated gel imaging system and analysed for calculation using ImageJ software. Main antibodies and dilution ratio: Anti-Akt (ab81283, Abcam, 1:1000); Anti-beta Amyoid 1-42 (ab201060, Abcam, 1:1000); Anti-IL-1beta (ab9722, Abcam, 1:800); Anti-IL-10 (ab9969, Abcam, 1:800); Anti-PI3K (ab191606, Abcam, 1:1000); Anti-GFAP (BA0056, BOSTER, 1:800); Anti-IBA-1 (PB0517, BOSTER, 1:800); Anti-Beclin1 (ab207612, Abcam, 1:1000); Anti-LC3 (ab192890, Abcam, 1:1000); Anti-p-Akt (ab8805, Abcam, 1:1000); Anti-β-actin (GB12001, Servicebio, 1:2000).
6
Protein Expression Analysis in Ovarian Tissue
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The homogenized ovarian tissues were lysed in RIPA lysis buffer containing a protease inhibitor. The supernatants obtained after centrifugation for 15 min at 13,000 rpm were then collected for western blotting. The protein concentration was detected by the Bradford test. Thereafter, the proteins were separated by Mini-PROTEAN® TGX™ (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes using a Trans-Blot Turbo Transfer System. The PVDF membranes were blocked with 5% horse serum at room temperature for 3 h and incubated at 4 °C overnight with rabbit polyclonal antibodies against caspase-3 (diluted 1:2000; ab184787, Abcam, Cambridge, UK), total PI3K (1:1000, ab191606, Abcam, Cambridge, UK), total AKT (1:500, ab8805, Abcam, Cambridge, UK), and total mTOR (1:800, Dg-Peptide Co., Hangzhou, China). Subsequently, the primary antibodies were incubated with the corresponding secondary antibodies (1:5000 sc-2357 and sc-516102 Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. The bands of the target proteins were imaged using a Bio-Rad Gel Documentation System and analyzed using Image Lab software (Bio-Rad, USA).
7
Western Blot Analysis of TGF-β1 Signaling
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Western blot analysis was performed as described by Rong et al. [30 (link)]. The basic steps included protein concentration, polyacrylamide gel electrophoresis, film rotation, closing, primary antibody incubation, film washing, secondary antibody incubation, film washing, and enhanced chemiluminescence (ECL) development. The antibodies were: anti-TGF-β1 (ab215715, Abcam), anti-α-SMA (ab7817, Abcam), anti-Smad2 (ab40855, Abcam), anti-Smad3 (ab40854, Abcam), anti-Smad7 (25840-1-AP, Proteintech), anti-PI3K (ab191606, Abcam), anti-p-Aktser473 (66444-1-Ig, Proteintech), anti-mTOR (ab134903, Abcam), anti-p-IkBα (ab133462, Abcam), anti-p-IKKβ (AF3010, Affinity), and anti-p-P65 (ab76302, Abcam).
8
Cell Proliferation Assay and Protein Expression
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A total of 5 × 103 cells/well of each group were seeded into 96‐well plates (Corning), cultured at 37°C for 12 h with 5% CO2, and then 10 μl CCK‐8 solution (Beyotime) was added to each well. The cells were then cultured at 37°C with 5% CO2 for another hour. Then the value of optical density (OD) in each group was evaluated 450 nM using Microplate ReaderM200 PRO (Thermo Fisher Scientific). For western blot analysis, the following antibodies were used: Phospho‐pan‐AKT (Ser473; Affinity), Phospho‐PTEN (S380; Affinity), GAPDH (AF7021; Affinity), anti‐PI3K (ab191606; Abcam) and anti‐AKT (ab18785; Abcam).
9
Protein Expression Analysis in Rat Hippocampus
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Proteins were extracted from rat hippocampal tissues using radio immunoprecipitation assay buffer (Beyotime) and quantified with a bicinchoninic acid assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts (20 µg) of tissue samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto the polyvinylidene fluoride membranes (Bio‐Rad, Hercules, CA, USA). After being blocked with 5% defatted milk, the membranes were incubated with primary antibodies against: Bax (ab32503, 1:1,000, Abcam), cleaved (C)-caspase3 (#9661, 1:1,000, Cell Signaling, Danvers, MA, USA), phosphorylated (p)-TrkB (ab229908, 1:1,000, Abcam), TrkB (ab187041, 1:5,000, Abcam), p-PI3K (ab182651, 1:1,000, Abcam), PI3K (ab191606, 1:1,000, Abcam), proBDNF (p1374, 1:400, Sigma–Aldrich), mBDNF (ab108319, 1:1,000, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (ab181602, 1:10,000, Abcam) at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab7090, 1:1,000, Abcam) at room temperature for 2 h. Eventually, protein bands were visualized with an enhanced chemiluminescence kit (Bio-Rad), and the intensity values were analyzed with C‐Digit software (LI-COR Biosciences, Lincoln, NE, USA).
10
Co-immunoprecipitation of ERBB2 and PI3K with ERBB3
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For the initial IP step, 10 µg anti-ERBB3 antibody or IgG control antibody (cat. nos. 4754 and 2729, respectively; Cell Signaling Technology, Inc.) was incubated with protein G magnetic beads according to the manufacturer's protocol (Pierce; Thermo Fisher Scientific, Inc.) in 150 mM NaCl/100 mM HEPES (pH 8.2) buffer for 2 h at 4°C to form the anti-ERBB3-protein G beads or anti-IgG-protein G beads, respectively. After washing, 300 µg cell lysate protein (obtained using M-PER™ Mammalian Protein Extraction Reagent; Pierce; Thermo Fisher Scientific, Inc.) was immunoprecipitated on a rotator overnight at 4°C using 20 µl antibody-protein G beads. The antibody-protein G beads were then collected by pulse centrifugation (5 sec at 14,000 × g) at 4°C. The antibody-protein G beads were washed with lysis buffer, SDS-sample buffer with dithiothreitol (50 mM) was added, and the samples were boiled for 4 min. The samples were electrophoresed on a 4–12% bis-tris gel followed by transfer to a PVDF membrane. Co-IP with ERBB2 or PI3K (p85) was assessed via WB, as aforementioned, using an anti-ERBB2 antibody or anti-PI3K (p85) antibody (1:1,000; cat. no. ab16901 and ab191606, respectively; Abcam).
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