For western blotting assays, adipose tissues were homogenized in lysis buffer (1% Triton X-100, 50 nM Tris HCl, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF) with protease cocktail (Roche) and phosphatase inhibitors (1 mM Na3Vo4, 10 mM NaF). Equal amounts of protein (30 μg) were separated by gel electrophoresis, transferred onto a PVDF membrane (Millipore, Billerica, MA), and incubated with primary antibodies against phosphorylated Smad3 (ab52903, Abcam, 1:1000), and total Smad3 (ab40854, Abcam, 1:2000). GAPDH (ab8245, Abcam, 1:4000) was used as a loading control. The protein in the membrane was visualized using a Bio-Rad Clarity Western ECL Substrate (1705060, Bio-Rad). Uncropped western blot images can be found in the Source Data file.
Ab52903
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab52903 is a monoclonal antibody that can be used for the detection of a specific target protein. The antibody is supplied in a buffer solution and is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ab40854, by Abcam (28 mentions)
Ab53100, by Abcam (9 mentions)
Ab215715, by Abcam (9 mentions)
Ab34710, by Abcam (8 mentions)
Ab40855, by Abcam (8 mentions)
Ab280888, by Abcam (7 mentions)
Ab181602, by Abcam (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Ab92486, by Abcam (5 mentions)
Ab8227, by Abcam (5 mentions)
Ab28379, by Abcam (5 mentions)
Ab33875, by Abcam (5 mentions)
Ab8226, by Abcam (5 mentions)
Ab8245, by Abcam (4 mentions)
Ab202445, by Abcam (4 mentions)
Ab2413, by Abcam (4 mentions)
Ab5694, by Abcam (4 mentions)
Ab188334, by Abcam (4 mentions)
110 protocols using ab52903
1
Adipose Tissue Phosphorylated Smad3 Analysis
2
Smad Signaling in Osteo-/Odonto-genesis
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Following 12-hr serum starvation, hBMSCs were cultured in osteo-/odonto-genesis induction medium (OIM) with or without 0.1, 1, 10 and 50 ng/mL rhTGFβ1, pH7.4 or pH10 dentin extracts, 700 ng/mL Noggin (R&D Systems) or 10 μM SB431542 (Selleckchem). After 30-min or 7-day culture, cells were washed with ice-cold PBS followed by protein extraction in RIPA Lysis Buffer (Thermo Scientific) with Protease/Phosphatase Inhibitor Cocktails (Cell Signaling Technology). Proteins were separated on a NuPAGE® Novex® 4–12% Bis-Tris Protein Gel (1.0 mm), transferred to nitrocellulose membrane (Bio-Rad), and detected with anti-phospho-Smad 2/3 (ab52903, 1:500, Abcam), anti-Smad 2/3 (ab202445, 1:500, Abcam), anti-Smad 1/5/9 (ab66737, 1:500, Abcam), anti-phospho Smad 1/5/8 (ab3848, 1:500, Millipore), anti-COL-1 (ab34710, 1:500, Abcam), anti-DSPP (sc-73632, 1:200, Santa Cruz Biotechnology), anti-RUNX2 (ab76956, 1:500, Abcam), anti-GAPDH (sc-25778, 1:200, Santa Cruz Biotechnology) antibodies. Images were developed with IR fluorescence & Odyssey using corresponding secondary antibodies (LI-COR).
3
Comprehensive Extracellular Matrix Analysis
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4
Protein Expression Analysis in Cellular Lysates
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Cell lysates were extracted using RIPA buffer (CW Biotech Co. Ltd., Beijing, China). The BCA protein assay kit (CW Biotech Co. Ltd., Beijing, China) was performed to measure the protein concentration. Equal amounts of proteins were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes and incubated with primary antibodies, including anti-SYK antibody (ab40781) (Abcam, Cambridge, MA, USA), anti-collagen I antibody (ab34710) (Abcam, Cambridge, MA, USA), anti-vimentin antibody (ab8978) (Abcam, Cambridge, MA, USA), anti-VEGF-A antibody (ab46154) (Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (ab1416) (Abcam, Cambridge, MA, USA), anti-α-SMA antibody, (ab32575) (Abcam, Cambridge, MA, USA), anti-TGF-β1 antibody (ab64715) (Abcam, Cambridge, MA, USA), anti-p-Smad3 antibody (ab52903) (Abcam, Cambridge, MA, USA), and anti-Smad3 antibody (ab40854) (Abcam, Cambridge, MA, USA). The membranes were then incubated in the secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1/1000) (ab6721) (Abcam, Cambridge, MA, USA) or anti-mouse immunoglobulin G secondary antibody (1/1000) (ab6785) (Abcam, Cambridge, MA, USA). The results were analyzed using the enhanced chemiluminescence (ECL) Western blot substrate (Pierce Biotechnology, Shanghai, China). GAPDH was used as an internal control.
5
Immunohistochemical Analysis of Signaling Pathways
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All the immunohistochemical assays were conducted following the manufacturer's instructions. Briefly, the skin tissues were fixed in formalin and embedded in paraffin. Consecutive paraffin sections (4-μm) of the tissue samples were prepared and incubated overnight at 4°C with primary antibodies followed by incubation with peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins (EnVision/HRP; Dako, Glostrup, Denmark). Primary antibodies against Smad3 (#9523), phospho-Stat3 (#9145), Stat3 (#12640), JAK1 (#3344), JAK2 (#3230) (dilution, 1:1,000; all from Cell Signaling Technology), phospho-Smad3 (ab52903), phospho-JAK2 (ab32101) (dilution, 1:1,000; both from Abcam), phospho-JAK1 (sc-101716; dilution 1:1,000; Santa Cruz Biotechnology, Inc.) were used.
6
Immunohistochemistry Assay for Fibrosis
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Antibodies against Fibronectin (ab2413), Collagen I (ab34710), α-SMA (ab5694), F4/80 (ab6640), CD4 (ab183685), CD8 (ab209775), p-Smad3 (ab52903) and pan Cadherin (ab51034) were from Abcam (Cambridge, UK). Antibodies against p-STAT3 (9145), STAT3 (9139), NF-κB p65 (8242), p-p65 (3033), p-AKT (4060), p-Smad2 (3108), Smad2 (5339), Smad3 (9523), p-Smad1/5/8 (13820), Smad1 (6944), Vimentin (5741), Tubulin (2148), rabbit IgGs (7074) and mouse IgGs (7076) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against IL-6 (21865), IL-6R (23457), AKT (10176), TGF-β (21898), gp130(21175) and Collagen Ⅲ (22734) were from Proteintech (Rosemont, IL, USA).
7
Quantitative Protein Analysis with Immunoprecipitation
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Cells were lysed by sonicating for 5 s in 1 ml of detergent-free lysis buffer (PBS, 5 mM EDTA, 0.02% sodium azide, 10 mM iodoacetamide, 1 mM PMSF, and 2 mg leupeptin) at 4°C. Antibody-conjugated beads were prepared by combining 1 mg of antibodies with 30 ml of a 50% protein A Sepharose bead slurry in 0.5 ml of ice-cold PBS for 1 hr at 4°C in a tube rotator and were then washed three times with 1 ml of lysis buffer. The antibodies used for coimmunoprecipitation were Akt (ab6076, abcam). The beads were washed three times with washing buffer (50 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA, 0.02% sodium azide, 0.1% Triton X-100) and once with ice-cold PBS. Immunodetection was carried out using the ECL Western Blotting Detection Kit (Amersham Corp). Proteins were detected using antibodies against Smad4 (ab40759, abcam), Pitx2 (sc-8748, Santa Cruz), p-Smad3 (ab52903, abcam), p-Smad2 (ab53100, abcam), B55α (ab185712, abcam), pAKT-T308 (ab5626, abcam), pAKT-S473 (ab8932, abcam) and GAPDH (ab9485, abcam). Relative protein expression levels were normalized to GAPDH levels.
8
Recombinant Protein-Mediated Cell Signaling
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Recombinant human TGF-β, recombinant FGF-2 and recombinant human TNF-α were purchased from Biolegend (San Diego, CA). Rapamycin was from ENZO life science (Loerrach, Germany). Bafilolycin A1 was from Invivogen. FITC-PHA was from Vector (FL-1111). FITC-PNA was from Sigma (L7381). DAPI was from Sigma (D9542). Alexa Flour 488, 594 or 647 conjugated anti-mouse or anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The primary antibodies against JLP (ab12331, 1:1000), Fsp-1 (ab197896, 1:200), α-SMA (ab124964, 1:1000), Fibronectin (ab45688, 1:500), Collagen-I (ab34710, 1:1000), Ki67 (ab16667, 1:250), TGF-β (ab92486, 1:500), phospho-Smad2 (ab188334, 1:1000), phospho-Smad3 (ab52903, 1:1000) and p62 (ab56416, 1:200) were all from Abcam (Cambridge, MA). Anti-LC3 antibody (ab51520, 1:100, Abcam) was used for immuno-staining and Anti-LC3 antibody (L7543, 1:1000, Sigma) was used for western blotting, respectively. Anti-Nephrin and anti-F4/80 (123120, 1:100) antibodies were from Progen and Biolegend, respectively. Anti-Caspase-3 (#9664, 1:1000), anti-Beclin 1 (#3738,1:1000) was from Cell signaling Technology (Danvers, MA). Anti-GAPDH (sc-365062, 1:2500) was purchased from Santa Cruz (Santa Cruz, CA).
9
Protein Extraction and Western Blot Analysis
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MSCs were lysed with RIPA buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Protein concentrations were determined with Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Proteins were separated on a 4–12% Bis-Tris gradient electrophoresis gel (Life Technologies, Carlsbad, CA) and transferred onto a nitrocellulose membrane. The membrane was then blocked in either 5% nonfat dry milk or 5% BSA followed by incubation with primary antibodies at 4°C overnight. Antibodies for pSmad3 (Abcam ab52903), Smad3 (Abcam ab40854), p-STAT3 (Cell Signaling 9145) and STAT3 (Cell Signaling 9139) were used. β–actin was used as loading control. Bands were quantified using Image J software (version 1.49).
10
Immunostaining of Zebrafish Embryos
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Embryos for most antibodies were fixed at the indicated stage in 4% paraformaldehyde, permeabilized in PBST (PBS+0.5% Triton X-100), and blocked in PBST+2% bovine serum albumin. Antibodies and concentrations were as follows: anti-Pax2a (GTX128127, Genetex; 1:200); anti-pSmad3 (ab52903, Abcam; 1:200); anti-Laminin 1 (L9393, Sigma-Aldrich; 1:100); anti-fibronectin (F3648, Sigma-Aldrich; 1:100); anti-GFP (A10262, Invitrogen; 1:200).
In accordance with methods described by Carrara et al. (2019) (link), anti-Nidogen 1/Entactin (ab14511, Abcam) was used at 1:100 and required a modified embryo preparation after 4% paraformaldehyde fixation: embryos were permeabilized in 30 μg/ml Proteinase K for 15 min, and blocked in 0.8% PBST+10% sheep serum+1% bovine serum albumin. The antibody was applied in 0.8% PBST+1% sheep serum+1% bovine serum albumin.
Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse (A-11001, Invitrogen), Alexa Fluor 488 goat anti-rabbit (A-11008, Invitrogen), Alexa Fluor 488 goat anti-chicken (A-11039, Invitrogen), all used at 1:200. Nuclei were detected by incubation with 1 µM TO-PRO-3 iodide (T3605, Invitrogen). Embryos were cleared through a series of 30%/50%/70% glycerol (in PBS) prior to imaging.
In accordance with methods described by Carrara et al. (2019) (link), anti-Nidogen 1/Entactin (ab14511, Abcam) was used at 1:100 and required a modified embryo preparation after 4% paraformaldehyde fixation: embryos were permeabilized in 30 μg/ml Proteinase K for 15 min, and blocked in 0.8% PBST+10% sheep serum+1% bovine serum albumin. The antibody was applied in 0.8% PBST+1% sheep serum+1% bovine serum albumin.
Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse (A-11001, Invitrogen), Alexa Fluor 488 goat anti-rabbit (A-11008, Invitrogen), Alexa Fluor 488 goat anti-chicken (A-11039, Invitrogen), all used at 1:200. Nuclei were detected by incubation with 1 µM TO-PRO-3 iodide (T3605, Invitrogen). Embryos were cleared through a series of 30%/50%/70% glycerol (in PBS) prior to imaging.
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