Antibodies for immunostaining and western blotting were used as per the manufacturer’s recommendations. The following antibodies were used within this study: β-tubulin (Cell Signalling Technology, Cat # 2146 (1:1000)), Cleaved Caspase-3 [Asp175] (Abcam, Cat # ab49899 (WB 1:500, IHC 1:1500)), CDKN2A/p19ARF (Abcam, Cat # ab80 (1:1000)), cMYC [Y69] (Abcam, Cat # ab32072 (WB 1:1000, IHC 1:500)), Cyclophilin B (Cell Signalling Technology, Cat # 43603 (1:1000)), GFAP [GA5] (Millipore, Cat # MAB3402 (1:1000)), GFP (Abcam, Cat # ab13970 (1:1000)), HSF1 (Abcam, Cat # ab61382 (1:1000)), HSP70 (Abcam, Cat # ab2787 (1:1000)), HSP90 [EPR16621] (Abcam, Cat # ab203085 (1:1000)), Ki67 [SP6] (Abcam, Cat # ab16667 (1:2000)), n-MYC [NCM II 100] (Abcam, Cat # ab16898 (1:250)), NPR3 (Abcam, Cat # ab97389 (1:250)), Olig-2 (Millipore, Cat # AB9610 (1:1000)), OTX2 (R&D Systems, Cat # AF1979 (1:500)), p21 (Abcam, Cat # ab109199 (1:1000)), Synaptophysin [SY38] (Millipore, Cat # MAB5258 (1:500)), TUJ1 (Covance, Cat # MMS-435P (1:500)), β-actin (Santa Cruz Biotechnology, Cat # sc-47778 (1:1000)), CDKN2A/p16INK4A (Abcam, Cat # ab211542 (1:1000)), anti-mouse IgG secondary HRP (VWR, Cat # NXA931 (1:1000)), anti-rabbit IgG secondary HRP (GE Healthcare, Cat # NA934 (1:1000)), and Lamin B1 (Abcam, Cat # ab16048 (1:1000)).
Cdkn2a p16ink4a
Manufactured by Abcam
Sourced in United States
CDKN2A/p16INK4a is a protein that plays a crucial role in cell cycle regulation. It functions as a tumor suppressor by inhibiting the activity of cyclin-dependent kinases, which are essential for cell cycle progression. This protein is involved in the regulation of cell proliferation and can contribute to the prevention of uncontrolled cell growth and cancer development.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (4 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Hsp70, by Abcam (1 mentions)
Olig2, by Merck Group (1 mentions)
C myc y69, by Abcam (1 mentions)
Cyclophilin b, by Cell Signaling Technology (1 mentions)
Gfap g a 5, by Merck Group (1 mentions)
Ki67 sp6, by Abcam (1 mentions)
N myc ncm 2 100, by Abcam (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Lamin b1, by Abcam (1 mentions)
Ab53034, by Abcam (1 mentions)
Ab54210, by Abcam (1 mentions)
Ab102842, by Abcam (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Pd0332991, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Pol 2, by Santa Cruz Biotechnology (1 mentions)
10 protocols using cdkn2a p16ink4a
1
Antibody Characterization for Immunostaining
2
Investigating Rb Pathway Regulation
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After serum starvation for 12 hours, cells were treated with increasing concentrations and exposure times of PD-0332991 (Sigma-Aldrich, St. Louis, MO, USA). Cells with or without treatment were washed twice with ice-cold phosphate-buffered saline, lysed, and separated into cytoplasmic and nuclear fractions using the Nuclear Extract Kit according to the manufacturer’s instructions (Active Motif, Carlsbad, CA, USA). To detect Rb, phospho-Rb, p15, p16, p18, p19 and cyclin D1 proteins, we separated the fractions using SDS polyacrylamide gel electrophoresis and electrotransferred the proteins to nitrocellulose membranes. Western blot analyses were performed with various specific primary antibodies including Rb (4H1, 9309, Cell Signaling Technology, Danvers, MA), phospho-Rb (Ser780, 8180, Cell Signaling), p15 (p15 INK4b, ab53034, Abcam, Cambridge, MA, USA), p16 (CDKN2A/P16INK4a, ab54210, Abcam), p18 (p18INK4c, ab3216, Abcam), p19 (p19 INK4d, ab102842, Abcam) and β-actin (4970, Cell Signaling). The immunoreactive bands in the immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit or mouse immunoglobulin using an enhanced chemiluminescence Western blotting system (ECL Plus, GE Healthcare Life Sciences, Pittsburgh, PA, USA). Nonspecific antigen sites were blocked with 10% bovine serum albumin in 1X Tris-buffered saline.
3
Immunodetection of Epigenetic Regulators
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Antibodies used were CTCF (Cell Signaling Technologies, 3418 for immunoblotting and immunoprecipitation), DDX5 (Abcam ab-21696), CDKN2A/p16INK4a (Abcam ab-108349), p14ARF (Santa Cruz Biotechnology sc-53639), Pol II (Santa Cruz Biotechnology sc-55492), GAPDH (Santa Cruz Biotechnology sc-32233), and histone H3 (Sigma-Aldrich H0164).
4
Modulation of Senescence Markers by D-panthenol
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hDPCs (1 × 106 cells/dish) were seeded in 100 mm culture dishes and cultured for 24 h. D-panthenol was treated at appropriate concentrations for 24 h. Cells were then washed with ice-cold PBS and lysed on ice in an M-PER buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a Complete™ protease inhibitor cocktail and phosphatase inhibitor (Roche, Indianapolis, IN, USA). 40 μg of protein was analyzed by Western blotting with appropriate antibodies to evaluate protein expression; β-catenin (1000:1 dilution, Santa Cruz, CA, USA), CDKN2A/p16INK4a (1000:1 dilution, Abcam, Cambridge, UK), p21 (1000:1 dilution, cell signaling technology, Danvers, MA, USA), caspase3 (1000:1 dilution, cell signaling technology, Danvers, MA, USA), GAPDH (2000:1 dilution, Santa Cruz, CA, USA). Western blot was analyzed by a chemiluminescence detector (iBright FL1500, Thermo Fisher Scientific, Waltham, MA, USA).
5
Protein Expression Analysis in Cells
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Twenty micrograms of total protein was used per sample. The following primary antibodies were used: γH2AX (1:1,000, 2577S; Cell Signaling Technology, Inc., Danvers, MA, USA); CDKN2A/p16ink4a (1:1,000, ab 81278; Abcam, Cambridge, UK); SIRT1 (1:1,000, ab 32441; Abcam); SIRT6 (1:1,000, ABE102; EMD Millipore, Billerica, MA, USA); anti-perilipin A (1:1,000, P1998-200UL; Sigma-Aldrich Co., St Louis, MO, USA); anti-MyoD1 (1:1,000; Abcam); cleaved caspase-3 (Asp175; 1:1,000, 9661S; Cell Signaling Technology, Inc.). GAPDH was used as a load control (1:5,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA). The optical densities were quantified using a densitometer and Image-Pro Plus.
6
Comprehensive Western Blot Protocol
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The protocol used for western blot has been previously described in detail [29 (link)]. In brief, cells were harvested, washed twice with PBS, and lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer. Lysates were boiled in 4× SDS-sample buffer and resolved on SDS-PAGE. SDS-PAGE-separated proteins were transferred by electrophoresis onto a nitrocellulose membrane. Finally, blots were probed with appropriate primary and appropriate secondary antibodies, as follows: p-Rb (9307; Cell Signaling Technology, Danvers, MA, USA), CCNE2 (11935-1-AP; Proteintech), p-ERK (SC-7383; Santa Cruz Biotechnology, Dallas, TX, USA), ERK (SC-154; Santa Cruz Biotechnology), FGFR2 (SC-6930; Santa Cruz Biotechnology), CDK2 (SC-6248; Santa Cruz Biotechnology), CDK4 (SC-23896; Santa Cruz Biotechnology), CDK6 (19117-l-AP; Proteintech), CDKN2A/p16 INK4a (ab16880; Abcam, Cambridge, UK) and β-actin (SC-47778; Santa Cruz Biotechnology). Densitometry readings/intensity ratio of each band. In addition, the whole blot showing all the bands with all molecular weight markers on the Western in the Supplementary Figure S5 .
7
Kidney Tissue Protein Extraction and Analysis
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Kidney tissue was minced in lysate buffer (150 mM NaCl, 50 mM Tris-HCL, 1 mM EDTA, and 2% SDS) plus phosphatase and protease inhibitors, sonicated, centrifuged at 18,800g for 10 minutes at 4°C, and reduced with dithiothreitol (DTT) (manufacturer information in Supplemental Table 1 ). Cells were lysed using MilliporeSigma’s Mammalian Lysis Buffer plus protease and phosphatase inhibitors, sheared using an insulin syringe, clarified by centrifugation at 18,800g for 10 minutes at 4°C, and quantified using the BCA protein assay (Thermo Fisher Scientific). Both cell and tissue proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with the following primary antibodies from Cell Signaling Technology: cleaved caspase 3 (9664S, clone 5A1E), pSTAT3 (9145S, clone D3A7), total STAT3 (12640S, clone D3Z2G), α-tubulin (DM1A, clone 3873S),MLKL (37705S, clone D6W1K), pRb (9308, Ser807, clone 811), and cyclinD1 (55506T, clone E3P5S). Additionally, the following antibodies were used: GAPDH (sc-25778, clone K0615, Santa Cruz Biotechnology Inc.); collagen I (AB765P, MilliporeSigma); RIP3 (2283, Pro Sci); CDKN2A/p16INK4a (ab211542, Abcam). Appropriate HRP-conjugated secondaries were used followed by ECL, and bands were detected by GeneGnome (Syngene) or ChemiDoc MP (Bio-Rad); they were quantified by ImageJ.
8
Immunohistochemical Analysis of Ovarian Tissue
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Unstained and paraffin-embedded ovarian tissue slides were deparaffinized with xylene and rehydrated with graded ethanol. For antigen retrieval, the slides were treated with Tris-EDTA (pH 9.0; Thermo Scientific) for 30 min at room temperature. The samples were blocked with hydrogen peroxide solution (Cat#: 88597, Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The slides were stained using a Polink-2 Plus HRP Broad Kit (Cat#: D41-18, GBI Labs, Bothell, WA, USA) with DAB (3,3′3diaminobenzidine), according to the manufacturer’s protocol. The slides were incubated at 4 °C overnight in a humid chamber with primary antibodies: γ-H2AX (1:500 dilution; Bethyl Laboratories, Montgomery, AL, USA), 8-OHdG (1:500, Cat#: sc-66036, Santa Cruz, Dallas, TX, USA), PARP (1:400, Cat#: 46D11, Cell Signaling Technology, Danvers, MA, USA), and CDKN2A/p16INK4a (1:100, Cat#: EPR20418, Abcam, Cambridge, MA, USA) in PBS containing 2% BSA. The slides were incubated for 1 h with the following secondary antibodies: goat anti-rabbit IgG (H + L), Alexa Fluor 488 conjugate (1:1000, Invitrogen, Waltham, MA, USA), goat anti-rabbit IgG (H + L), and Alexa Fluor 594 conjugate (1:1000, Invitrogen). The slides were counterstained with Mayer’s hematoxylin (Cat#: HMM125, Scytek, West Logan, WV, USA) and antifade mounting medium with DAPI (Cat#: H-1200, Vector Laboratories, Burlingame, CA, USA).
9
Immunohistochemical Analysis of Embryonic Tissues
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Embryos were fixed in 4% PFA overnight, embedded into OCT compound, and frozen. Frozen samples were sectioned at 5 μm thickness, dried up on slide glass, fixed in 4% PFA, and endogenous peroxidase activity was quenched by incubating the slides with 0.3% hydrogen peroxide. The sections were subjected to antigen retrieval by microwaving in sodium citrate (0.01 M, pH 6.0) as the antigen unmasking solution and then incubated overnight at 4 °C with the primary antibody. Antibodies used for this experiment are as follows: PCNA (PC 10, monoclonal, Sigma-Aldrich, St Louis, MO, USA), p27 (mouse monoclonal, Cell Signaling Technology, Danvers, MA, USA), activated caspase-3 (Cell Signaling Technology), BrdU (Bu20a, mouse monoclonal, Cell Signaling Technology), Nestin (clone 401, monoclonal, Merck Millipore), Hes5 (AB5708, polyclonal, Merck Millipore), and CDKN2A/p16INK4a (2D9A12, monoclonal, abcam).
Immunostaining with the mouse monoclonal antibody was carried out using an ABC kit (Vector Laboratories, Burlingame, CA, USA). After incubation with antibodies, sections were washed three times in PBS and then incubated with the appropriate biotinylated secondary antibody (DAKO and Vector Laboratories) followed by StreptABComplex/HRP (DAKO and Vector Laboratories) following the manufacturer’s instructions.
Immunostaining with the mouse monoclonal antibody was carried out using an ABC kit (Vector Laboratories, Burlingame, CA, USA). After incubation with antibodies, sections were washed three times in PBS and then incubated with the appropriate biotinylated secondary antibody (DAKO and Vector Laboratories) followed by StreptABComplex/HRP (DAKO and Vector Laboratories) following the manufacturer’s instructions.
10
Quantitative Analysis of Cellular Signaling
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Total RNA was extracted using TRIzol reagent (Invitrogen) according to instruction. The purity of RNA was tested by measuring the absorbance (NanoDrop ND‐1000, Thermo Fisher Scientific) and reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara). The expressions of genes were measured with Bio‐Rad iQ5 real‐time PCR detection system, with β‐actin for normalization. The primers for target genes were listed in Table 1 .
Protein was extracted by whole cell lysis assay (KeyGEN), and the concentrations were evaluated by enhanced BCA protein assay kit (Beyotime). Equal amount of protein samples was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio‐Rad Laboratories) and transferred onto a polyvinylidene fluoride membrane (Millipore). Following incubation in specific antibodies, we probed proteins by enhanced chemiluminescence kit (Bio‐Rad Laboratories). Bands were quantified by ImageJ software. Specific antibodies included CREB (Assay biotechnology, 1:1000), pCREB (Assay biotechnology, 1:1000), CDKN2A/p16INK4a (Abcam, 1:1000), pSMAD3 (Abcam, 1:1000) and Tubulin α (Signalway Antibody, 1:2000).
Protein was extracted by whole cell lysis assay (KeyGEN), and the concentrations were evaluated by enhanced BCA protein assay kit (Beyotime). Equal amount of protein samples was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio‐Rad Laboratories) and transferred onto a polyvinylidene fluoride membrane (Millipore). Following incubation in specific antibodies, we probed proteins by enhanced chemiluminescence kit (Bio‐Rad Laboratories). Bands were quantified by ImageJ software. Specific antibodies included CREB (Assay biotechnology, 1:1000), pCREB (Assay biotechnology, 1:1000), CDKN2A/p16INK4a (Abcam, 1:1000), pSMAD3 (Abcam, 1:1000) and Tubulin α (Signalway Antibody, 1:2000).
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