Cells and muscle tissue were lysed in RIPA buffer (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. Protein lysates were heated at 95°C for 5 min in 5 × SDS sample buffer, then separated by 10% SDS polyacrylamide gel electrophoresis (30 μg each lane), followed by transfer to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, United States) using a Mini Trans-Blot Cell system (Bio-Rad). The membrane was blocked with 5% non-fat milk for 3 h. The primary antibodies were incubated overnight at 4°C. The membranes were washed and incubated with secondary antibodies for 1 h at 37°C followed by visualization by enhanced chemiluminescence (Bio-Rad). Primary antibodies specific for EZH2 (ab3748, 1:1,000; Abcam, Cambridge, United Kingdom), MyoD (sc-760, 1:1,000; Santa Cruz Biotechnology, Dallas, TX, United States), MyoG (sc-12732, 1:200; Santa Cruz Biotechnology), MyHC (sc-376157, 1:3,00; Santa Cruz Biotechnology), Ki67 (ab16667, 1:1,000; Abcam), p21 (sc-6246, 1:1,000; Santa Cruz Biotechnology), and β-actin (sc-4777, 1:1,000; Santa Cruz Biotechnology), along with goat anti-mouse IgG-HRP (sc-2005, 1:3,000; Santa Cruz Biotechnology) and goat anti-rabbit IgG-HRP (sc-2004, 1:3,000; Santa Cruz Biotechnology) secondary antibodies were used to detect protein expression.
Ab3748
Manufactured by Abcam
Sourced in United Kingdom
Ab3748 is a lab equipment product offered by Abcam. It is a device used for scientific research and analysis purposes. The core function of this product is to [insert concise, factual description of the product's core function without any interpretation or extrapolation].
Automatically generated - may contain errors
Lab products found in correlation
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (5 mentions)
Ab6002, by Abcam (4 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab1765, by Abcam (2 mentions)
Magnetic rna protein pull down kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab16667, by Abcam (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Mini trans blot cell system, by Bio-Rad (1 mentions)
Sc 12732, by Santa Cruz Biotechnology (1 mentions)
Sc 376157, by Santa Cruz Biotechnology (1 mentions)
Sc 4777, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ab3580, by Abcam (1 mentions)
Ab32644, by Abcam (1 mentions)
Ab1801, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
19 protocols using ab3748
1
Western Blot Analysis of Muscle Proteins
2
Investigating Protein-RNA Interactions
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The RNA immunoprecipitation (RIP) experiments were performed using the Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (17‐700, Millipore) following the manufacturer's instructions. After incubation of the microglial lysate with the anti‐EZH2 (ab3748, Abcam) or anti‐GR (ab3580, Abcam) or anti‐RbAp48 (ab1765, Abcam) or anti‐RING1A/B (ab32644, Abcam) antibody, the co‐precipitated RNA was detected by qPCR. The ratio of RNA binding with a specific antibody versus the total RNA was calculated.
3
Histone Modification Immunoblotting Protocol
4
Chromatin Immunoprecipitation Assay Protocol
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Chromatin immunoprecipitation (ChIP) assay was performed using ChIP Kit (Millipore, 17–371) according to the manufacturer’s instructions. Each ChIP reaction was performed using 1 μg of antibodies against Ezh2 (Abcam, ab3748), H3k27me3 (Abcam, ab6002) or IgG applied as negative control. Fold enrichment was quantified using qPCR. All promoter primers were listed in Supplementary Table S3 .
5
ChIP Assay for Histone Modifications
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ChIP assays were performed using a Magna ChIP kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The primers used for the ChIP assays are listed in the Supplementary Table S4 . MacroH2A1 (07–219, Millipore), p300 (SC-584, Santa Cruz), EZH2 (ab3748, Abcam), SUV39H1 (ab12405, Abcam), H3K27ac (ab4729, Abcam), H3K27me3 (ab6002, Abcam), and H3K9me3 (ab8898, Abcam) antibodies were used to immunoprecipitate chromatin fragments.
6
RNA Immunoprecipitation for Studying Protein-RNA Interactions
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RIP was performed as previously described (Ng et al., 2012 (link)). In each immunoprecipitation for the RIP assay, 3 μg of the appropriate antibody was used. For the exogenous RIP assay, an anti-Sox2 (Abcam, ab59776) or anti-Nanog (Abcam, ab80892) antibody was used to bind to the RNA following ectopic expression of linc1614 with Sox2 or Nanog in 293T cells. For the native RIP assay, an anti-Sox2, anti-Nanog, anti-Oct4 (Abcam, ab19857), anti-Ezh2 (Abcam, ab3748), or anti-Suz12 (Abcam, ab12073) antibody was used to pull down the RNA associated with the corresponding proteins. The immunoprecipitated RNA was extracted using RNAiso and analyzed via qPCR. The fold enrichment was calculated relative to the corresponding control IgG sample.
7
RIP Assay for EZH2 Protein
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RIP experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. Antibody for RIP assays of EZH2 was from Abcam (ab3748). When RIP treated with RNase, lysates were incubated with RNase for 1 hours in 37 °C. The co-precipitated RNAs were detected by conventional RT-PCR and real-time PCR. The primer sequences are listed in Supplementary Table 1 .
8
Protein Extraction and Quantification
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The cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors to extract proteins. The protein samples were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the membranes were incubated with the primary antibodies. Anti-PGC-1α (sc13067, Santa Cruz, 1:1000), anti-EZH2 (ab3748, Abcam, 1:1000), and anti-β-actin (A5441, Sigma-Aldrich, 1:2500) antibodies. The protein bands were analyzed using Image Lab software (Bio-Rad), and the gray values of experimental proteins were detected using ImageJ software.
9
ChIP-qPCR Reverse Cross-Linking Protocol
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Chips were performed as described in [8 (link)]. Briefly, ChIP material was reverse cross-linked at 65 °C for 4 h, purified using PCR purification kit (Invitrogen, USA) and used for enrichment analysis using primers listed in Additional file 1 . Antibodies used were same as for westerns except for EZH2 (ab3748, Abcam).
10
RIP Assay for Studying Protein-RNA Interactions
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RIP experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. HXT116 and SW480 cells were lysed in complete RIP lysis buffer, and the cell extract was incubated with magnetic beads conjugated with specific antibodies or control IgG (Millipore) for 6 hr at 4°C. Beads were washed and incubated with Proteinase K to remove proteins. Finally, purified RNA was subjected to real-time qPCR analysis. Antibody for RIP assays of EZH2 was from Abcam (ab3748).
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