Transfection reagents Lipofectamine 2000 and Lipofectamine 3000 were purchased from Invitrogen, RNA synthesis inhibitor actinomycin D, protease inhibitor MG132, lysosomal inhibitors bafilomycin and hydroxychloroquine, CDK inhibitor RO-3306, and thymidine were purchased from Sigma-Aldrich. λPPase was purchased from New England BioLabs. Anti-CELF6 (ab173282) and anti-GFP (ab1218) were obtained from Abcam, anti-p21Waf1/Cip1 (#2947), anti-p27Kip1(#3686), anti-Gadd45α (#4632), anti-Cyclin B1(#12231), anti-β-TrCP (#4394), anti-Wee1 (#13084), anti-cleaved PARP (#5625), anti-cleaved Casp3 (#9661), anti-cyclin E1 (#20808), and anti-Ubiquitin (#3936) antibodies were purchased from Cell Signaling Technology, anti-P53 (10442-1-AP), anti-GFP (66002-1-1 g), anti-His (66005-1-1 g), anti-β-tubulin (60008-1-1 g), and anti-GAPDH (10494-1-AP) were obtained from Proteintech, anti-LC3B (L7543) was obtained from Sigma-Aldrich.
Anti p27 kip1
Manufactured by Abcam
Sourced in United States
The Anti-p27 Kip1 antibody is a research tool used to detect the p27 Kip1 protein, a cyclin-dependent kinase inhibitor that plays a role in cell cycle regulation. This antibody can be used in various immunoassay techniques to identify and quantify the presence of p27 Kip1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (3 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (3 mentions)
Anti cleaved caspase 3, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Odyssey v2, by LI COR (3 mentions)
Anti cyclin e1, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti cdk2, by Abcam (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Mg132, by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
Ro 3306, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Bafilomycin, by Merck Group (1 mentions)
Anti cyclin b1, by Cell Signaling Technology (1 mentions)
Anti cyclin e1, by Cell Signaling Technology (1 mentions)
Anti wee1, by Cell Signaling Technology (1 mentions)
Hydroxychloroquine, by Merck Group (1 mentions)
10 protocols using anti p27 kip1
1
Cell Signaling Protein Analysis Protocol
2
Western Blot Analysis of HER2 Signaling
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A 20 μg protein aliquot was resuspended in Laemmli buffer, resolved on polyacrylamide gels under reducing conditions and transferred to polyvinylidine difluoride membranes (Immobilon-P, EMD Millipore Corporation). Membranes were blocked with 5% skim milk or 5% BSA in Tris buffer saline (TBS) with 0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature, and incubated with appropriate primary antibodies: anti-phospho-HER2/ErbB2 Tyr1248 (Cell Signaling Technology, Inc.), anti-HER2/ErbB2 (clone 29D8, Cell Signaling Technology, Inc.), anti-p27 KIP1 (Abcam), or anti-α-tubulin (Sigma-Aldrich). Antibodies conjugated to horseradish peroxidase (Abcam) were used as secondary antibodies, and chemiluminescence reaction was developed with the ECL star kit (Euroclone). Densitometric analysis of protein bands was performed with ImageJ software.
3
Hyperoxia Regulation of Cell Cycle Proteins
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The cells were grown in complete medium on six-well plates to 70–80% confluence, after which they were exposed to normoxia (95% air and 5% CO2) or hyperoxia (95% O2 and 5% CO2) for up to 48 h. In a separate set of experiments, the cells grown on six-well plates were treated with DMSO or 30 µM PD98059 for 24 h. Following these treatments, whole-cell protein extracts were obtained by using the radio immunoprecipitation assay lysis buffer system (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-24948) and subjected to western blotting with the following antibodies: anti-β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-47778, dilution 1:1000), anti-cyclin A (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-751, dilution 1:1000), anti-cyclin D1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-8396, dilution 1:250), anti-cyclin-dependent kinase (Cdk) 4 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-23896, dilution 1:250), anti-p27 Kip 1 (Abcam, Cambridge, MA, USA ; ab32034, dilution 1:1000), anti-total ERK1/2 (Cell Signaling, Danvers, MA, USA; 4695, dilution 1:1000), anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA; 9106, dilution 1:1000). The immunoreactive bands were detected and quantified as described in the “in vivo experiments” section.
4
Western Blot Analysis of Protein Expression
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Total protein was isolated from cell lysates or tumor tissues with RIPA buffer, and quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and then transferred into PVDF (Bio-Rad) membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Then, membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. After that, membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study were as follows: anti-MAGOH (Abcam, Cambridge, MA, USA; 1:1000), anti-MAGOHB (Abcam; 1:1000), anti-ERK (Abcam; 1:1000), anti-p-ERK (Abcam; 1:1000), anti-mTOR (Abcam; 1:1000), anti-RAF-b (Abcam; 1:1000), anti-MEK (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Cyclin B1 (Abcam; 1:1000), anti-p27 Kip1 (Abcam; 1:1000) and anti-CDK1 (Abcam; 1:1000). GAPDH was used as an internal control.
5
Antibody Production and Characterization
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Psmd14 and P97 antisera were produced by immunizing rats and rabbits against full-length mouse Psmd14 and rat P97, respectively. GFP antiserum was produced by immunizing rabbits against full-length GFP. Antibodies and their sources were anti-Psma1 (ABR, Golden, CO) or monoclonal antibody 2-17 (a kind gift from Keiji Tanaka, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), anti-ubiquitin (Zymed-Invitrogen Waltham, MA), anti-Rpt1 (Biomol, Kelayres, PA), anti-Flag M2 (Sigma-Aldrich); anti-S5a (Abcam, Cambridge, UK; and a CPTC-PSMD4 hybridoma [52242.DSHB] obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), anti-hemagglutinin (HA; Covance, Princeton, NJ), anti-hPlic (Abcam), anti-P27kip1 (Abcam), anti-T7 tag (ICL, Portland, OR), and anti-tubulin (Sigma-Aldrich). Where shown, Western blotting quantifications were performed by ImageJ software (National Institutes of Health, Bethesda, MD) on at least three independent experiments.
6
Western Blot Analysis of Cell Signaling
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Total protein was isolated from cell lysates by using RIPA buffer, and quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and then transferred onto PVDF (Bio-Rad) membranes. After blocking with 5% skim milk in TBST for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study were as follows: anti-p27 Kip1 (Abcam, Cambridge, MA, USA; 1:1000), anti-Bax (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Bcl-2 (Abcam; 1:1000), anti-cleaved caspase 9 (Abcam; 1:1000), anti-CDK2 (Abcam; 1:1000), anti-Cyclin E1 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.
7
Organoid Immunostaining and Imaging
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For staining and imaging, organoids were allowed to adhere ON onto transwell-permeable support membranes (Corning) in 12-well plates coated with 100% Matrigel. The next day, they were fixed with 4% PFA (10 min). Cells were permeabilized with 3% Triton-X 100 for 10 min and incubated ON with blocking buffer containing 2% BSA and 0.01% Triton-X 100. Primary antibodies were incubated ON at 4°C (1:200 dilution). Anti-Sox2 (mouse, BD Bioscience), anti-Pou4f3/Brn-3c (mouse, Santa Cruz Biotechnology) anti-Myo7a (rabbit, Enzo Life Sciences) and anti-p27/Kip1 (rabbit, Abcam). Organoids were then rinsed and incubated ON with the secondary antibodies as described above. For FM1-43 uptake, organoids were first incubated with Hoechst (10 μg/ml), 30 min at 37°C, then a brief incubation FM1-43 (5 μM; Life Technologies) for 30 s, and washed twice before fixation (4% PFA). To image organoids, inserts were placed face down in a 24 well glass bottom plate (MatTek) and imaged on a Zeiss LSM710 laser scanning microscope (5 μm Z-stacks intervals). Compiled Z-projections were processed (FIJI, ver2.0) and hair cells quantified on individual planes with manual cell counter plugin, by evaluation of cytosolic expression and nuclear exclusion of Myo7a. Organoid volumes were estimated from the sum of the product of the area of the organoid (at each plane) and the Z stack height.
8
Western Blot Analysis of Cellular Proteins
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Western blotting was performed as previously described41 (link). Briefly, whole cellular lysates were prepared by harvesting the cells in cell lysis buffer. The antibodies used were purchased from Santa Cruz Biotechnology (anti-DNMT1, anti-P-gp), Cell Signaling Technology (anti-p27kip1), Abcam (anti-nucleolin) and Sigma Aldrich (anti-β-actin).
9
Profiling RAS/MAPK Signaling in Liver
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Liver tissues were homogenized and digested in 1× RIPA buffer containing phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA). Western blot experiments were performed following the standard protocol. The following primary antibodies were used: anti-Pan-RAS (sc-14022; Santa Cruz Biotechnology), anti-phospho-AKT (#4060, Cell Signaling Technology), anti-phospho-MEK (#9154, Cell Signaling Technology), anti-phospho-ERK (#4370, Cell Signaling Technology), anti-p21Cip1 (ab2961; Abcam), anti-p27Kip1 (ab7961; Abcam), anti-p16INK4A (10883-1-AP; Proteintech), anti-HP1γ (ab10480, Abcam), anti-DcR2 (ab2019; Abcam), and anti-GAPDH (#2118; Cell Signaling Technology). Anti-rabbit IgG–HRP (Sigma-Aldrich) was used as the secondary antibody. Bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
10
Western Blot Analysis of Cell Signaling
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Total protein was isolated from tissue or cell lysates by using RIPA buffer and quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and transferred to PVDF (Bio-Rad) membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. After that, membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study: anti-CDK2 (Abcam, Cambridge, MA, USA; 1:1000), anti-cyclin E1 (Abcam; 1:1000), anti-p27 Kip1 (Abcam; 1:1000), anti-cleaved caspase3 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.
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