Huh7 cells were lysed in NET buffer (50 mM Tris-HCl [pH 7.0], 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40) with phosphatase inhibitors and cocktail protease inhibitors. Lysate was incubated on ice for 30 min, and cleared by centrifugation at 13,000 x g for 30 min at 4 °C. IP was performed with rabbit polyclonal Smad3 Ab (Abcam, ab28379) in the presence of protein A Sepharose (Dynabeads Protein A, #10002D, Invitrogen; USA) for 2 h at 4 °C in a rocking incubator. The Smad3 antibody from Abcam (ab28379) was previously validated using Smad3 KO animals and the data have been published [23 (link)]. Resulting immunocomplexes were subjected to immunoblotting. Blots were probed with anti-HDV-positive human serum to detect L-HDAg or S-HDAg, then incubated with HRP-conjugated goat anti-human IgG + IgM secondary Ab for 1 h. Bands were visualized with ECL reagents (PerkinElmer).
Ab28379
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab28379 is a monoclonal antibody that detects the CD4 antigen. CD4 is a glycoprotein expressed on the surface of T helper cells, regulatory T cells, and monocytes. This antibody can be used in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52903, by Abcam (5 mentions)
Ab92486, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Ab4729, by Abcam (2 mentions)
Nf κb p65, by Santa Cruz Biotechnology (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Amersham ecl prime detection reagent, by GE Healthcare (2 mentions)
Ab32360, by Abcam (2 mentions)
Sc 225, by Santa Cruz Biotechnology (2 mentions)
α tubulin sc 69969, by Santa Cruz Biotechnology (2 mentions)
Sc 8008, by Santa Cruz Biotechnology (2 mentions)
Amersham ecl prime, by Cytiva (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab5694, by Abcam (2 mentions)
Ab40759, by Abcam (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Truseq chip library preparation kit, by Illumina (1 mentions)
Sc 899, by Santa Cruz Biotechnology (1 mentions)
36 protocols using ab28379
1
Immunoprecipitation and Immunoblotting of Smad3
2
Chromatin Immunoprecipitation and Analysis
3
Western Blot Analysis of T Cell Signaling
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Following cytokine stimulations, proteins were extracted from CD4+ T cells and separated into cytosol and nuclear fractions using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instruction. The samples were separated on 8 or 10 % SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with blocking buffer (TBS containing 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk). After 3 washes with TBS-T, the membrane was incubated overnight with primary antibodies at 1: 1,000–1: 4,000 dilutions in blocking buffer at 4 °C. After 3 washes with TBS-T, the membrane was incubated with corresponding secondary antibody at 1: 4,000 dilution in blocking buffer for 1 h. After 3 washes with TBS-T, the proteins were visualized using Amersham ECL Prime Detection Reagent (GE Healthcare). The primary antibodies used were: α-Tubulin (sc-69969, Santa Cruz); NF-κB p65 (sc-8008, Santa Cruz); NF-κB p105/p50 (ab32360, Abcam); SMAD3 (ab28379, Abcam); TFIIB (sc-225, Santa Cruz). The secondary antibodies used were: anti-mouse IgG (#7076, Cell Signaling Technology); anti-rabbit IgG (#7074, Cell Signaling Technology).
4
Quantitative Western Blot Analysis
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Western blot analysis was performed as previously described (Ochs et al., 2013 (link)). Briefly, the Odyssey Imaging System (Li-COR Biosciences) was used which allows a linear quantification using near-infrared fluorescence. The membranes were incubated with primary antibodies that recognize CUGBP1 (ab9549, Abcam), SMAD2 (sc-6200, Santa Cruz), SMAD3 (ab28379, Abcam), SMAD4 (ab3219, Abcam), p38 (sc-535, Santa Cruz), NDUFS2 (ab96160, Abcam), mPGES-1 [160140, Cayman-Chemical (Westman et al., 2004 (link))], β-Actin (sc-1616, Santa Cruz).
5
Quantifying Protein Levels in MEFs
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MEFs grown on six wells were harvested in lysis buffer containing 25 mM Tris-HCl, pH 7.5, 300 mM NaCl and 1% Triton with protease and phosphatase inhibitors. Tissues were minced by a Dounce homogenizer using 1-2 ml RIPA buffer. A total of 30 µg of protein were separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking of the membranes by 5% dry milk/TTBS, membranes were incubated in following primary antibody solutions: anti-Smurf1 (Novus, 1D7); anti-Smurf2 (Abcam, EP629Y3); anti-Smad1 (Cell Signaling, 9743); anti-Smad2 (Abcam, EP784Y); anti-Smad3 (Abcam, ab28379), anti-Smad5 (Abcam, EP619Y), anti-phospho-Smad1/5 (Cell Signaling, 41D10), anti-phospho-Smad2 (Cell Signaling, 138D4); anti-phospho-Smad3 (Rockland, 600-401-919), GAPDH (Santa Cruz, 0411), HSC70 (Santa Cruz, B-6). Protein detection was carried out using HRP-coupled species-specific secondary antibodies and ECL solution, exposed to Hyperfilm ECL.
6
Immunohistochemical Analysis of Inflammatory Markers
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Six slides, in the order of one every 15 consecutive slides, from each mouse were subject to H&E staining to screen sections with most prominent inflammatory responses. Immunohistochemistry was performed on the properly selected consecutive frozen slides using VECTASTAIN Elite ABC Kit (Vectorlabs). Briefly, air-dried slides were fixed in methanol containing 0.3% H2O2 for 10 minutes and serum blocked for 20 minutes. The following incubation steps of primary antibody, secondary antibody, ABC reagent and DAB substrate were performed according to the manufacturer’s protocol. The slides were counterstained with hematoxylin. The primary antibodies (1:50 dilution) against procollagen COL1A2 (Santa Cruz, sc-8787), SRF (Santa Cruz, sc-335) and SMAD3 (Abcam, ab28379) were used. The secondary antibodies without the primary antibodies were used as the negative control.
7
ChIP Assay for Transcription Factor Binding
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ChIP assay was performed as previously reported (42 (link)). Primary HSCs were cross-linked by 1.42% formaldehyde followed by quenching with 125 mM glycine. Cells were lysed by ChIP buffer with protease inhibitor cocktail. After sonication, sheared chromatin supernatants were added with normal rabbit immunoglobulin G (IgG) (2729, Cell Signaling Technology), anti-Stat5 antibody (94205, Cell Signaling Technology), or anti-Smad3 antibody (ab28379, Abcam) followed by precipitation with Protein A Magnetic Beads (S1425S, New England Biolabs). The DNA was eluted from the beads and heated to reverse cross-linking before being subjected to real-time PCR using the following ChIP primers (43 (link)): Acta2: 5′-CAAGTCCTCAGCTAATGGCC-3′ (forward) and 5′-GGGGATAAACATCCTAAGCC-3′ (reverse); Col1a1: 5′-CCTCTGCCTCTTCTTGAGAGC-3′ (forward) and 5′-GGAGAGGAGCTAAGTGTGAAGC-3′ (reverse); Glrx promoter: 5′-GTACCCACCTTACAGGGCAA-3′ (forward) and 5′-TGCATAGTGATTGGGCCTTG-3′ (reverse); Bcl2 promoter: 5′-TTGCCGAGAAGAAGGGAGAA-3′ (forward) and 5′-CGGCGGCAGATGAATTACAA-3′ (reverse); Il10 promoter: 5′-ATTGTAAAACAGGGCCATGG-3′ (forward) and 5′-GGCAGTTGGTCAGAGGAGAG-3′ (reverse); Cdkal1 promoter: 5′-GGCAAATGGATCAGTGCTCA-3′ (forward) and 5′-TCCAAACTGCGAGAACAAGC-3′ (reverse).
8
TGF-β Signaling Pathway Regulation
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Recombinant TGF-β (TGF-β3) and the TGF-β type I receptor inhibitor SB431542 were purchased from R&D systems and Sigma-Aldrich (S4317), respectively. The following antibodies were used: mouse anti-FLAG (M2; Sigma-Aldrich), mouse anti-myc (9E10; Oncogene research products), rabbit anti-pSmad3 (C25A9; Cell Signaling), mouse anti-tubulin (DM1A; Sigma-Aldrich), rabbit anti-HDAC1 (2E10; Millipore), mouse anti-Smad2/3 (BD), rabbit anti-Smad3 (ab28379 and ab40854; Abcam), mouse anti-Smad4 (B-8; Santa Cruz), goat anti-Smad4 (AF2097; R&D) and mouse anti-TTF-1 (8G7G3/1; Novus Biologicals).
9
Signaling Pathways Modulated by MSTN Inhibition
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To assess the effects of MSTN inhibition on downstream pathways, we assayed for Akt/mTOR and Smad2/3 signaling, with blinding to treatment groups. Briefly, triceps muscle homogenates were heated at 65°C for 30 min, separated on 10% SDS–PAGE gels (20 µg of proteins), and transferred at 4°C to PVDF membranes (Immobilon-FL) (Goh and Millay, 2017 (link)). Membranes were subsequently blocked in 5% BSA/TBS-Tween, and incubated overnight at 4°C with an antibody against phosphorylated Akt (Ser473) (1:750; Cell Signaling #9271), total Akt (1:750; Cell Signaling #9272), phosphorylated Smad2 (Ser465/467) (1:1000; Cell Signaling #3108), total Smad2 (1:1000; Cell Signaling #5339), phosphorylated Smad3 (Ser423, Ser425) (1:2000 Abcam #ab51451), or total Smad3 (1:2000; Abcam #ab28379). GAPDH (1:5000; Cell Signaling #2118) served as a control for sample loading. Membranes were then washed and incubated with a DyLight anti-rabbit secondary antibody (1:5000; Cell Signaling #5151). Western blot signals were subsequently imaged and the relative abundance of phosphorylated and total protein levels of Akt, Smad2, and Smad3 were quantified as described above.
10
ChIP-seq analysis of Huntington's disease
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We prepared duplicate ChIP samples for each antibody from 4‐month‐old HttQ111/+ and from age‐matched wild‐type mice. For each ChIP preparation, chromatin DNA was prepared using the combined striatal tissue from both hemispheres of three mice. Preliminary experiments suggested that this was the minimal amount of material required to provide enough material for multiple IPs. Striata were transferred to a glass dounce on ice and homogenized in cold PBS with protease inhibitors. High‐resolution X‐ChIP‐seq was performed as described (Skene et al, 2010 ), with slight modifications. IPs were performed using Abcam anti‐SMAD3 antibody ab28379 [ChIP grade] or anti‐RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] [ChIP Grade] ab817. Sequencing libraries were prepared from the isolated ChIP DNA and from input DNA controls as previously described (Orsi et al, 2015 ). Libraries were sequenced on an Illumina HiSeq 2500 sequencer to a depth of ~17–25 million paired‐end 25‐bp reads per sample. Sequence reads have been deposited in GEO, accession GSE88775.
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