Cells treated with DMSO, Niebla sp. (1), or tumidulin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [24 (link)]. Antibodies against ALDH1, CD44, CD133, Lgr5, Msi1, and α-tubulin were used as previously described [17 (link)]. Antibodies against Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), and β-Actin (sc-47778; SANTA CRUZ) were detected with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) using an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA) and luminescence imaging on an Image Quant LAS 4000 mini. Bands were measured using Multi-Gauge 3.0 software, and the relative density was calculated based on the density of the β-actin bands in each sample. Values are expressed as arbitrary densitometric units corresponding to signal intensity.
Ab72130
Manufactured by Abcam
Sourced in United States
Ab72130 is a laboratory equipment product. It is a primary antibody that can be used in various immunological techniques. The core function of this product is to specifically bind and detect a target molecule in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hrp substrate kit, by Merck Group (2 mentions)
Sc 20687, by Santa Cruz Biotechnology (2 mentions)
Sc 271786, by Santa Cruz Biotechnology (2 mentions)
Ab53715, by Abcam (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini, by Merck Group (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Ab134906, by Abcam (1 mentions)
Ab53281, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab38295, by Abcam (1 mentions)
Ab52865, by Abcam (1 mentions)
Ab75850, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Sc 166362, by Santa Cruz Biotechnology (1 mentions)
Cd133, by Cell Applications (1 mentions)
Goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
12 protocols using ab72130
1
Protein Expression Analysis in Cancer Cells
2
Hedgehog Signaling Pathway Protein Analysis
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RIPA containing PMSF and phosphotransferase inhibitors (Beyotime Technology, Jiangsu, China) was used for protein extraction. Protein concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equivalent protein concentrations were separated using SDS‐PAGE (12. 5%) and transferred onto PVDF membranes, which were subsequently blocked with 5% nonfat milk and incubated with relevant primary antibodies at 4°C overnight. Horseradish peroxidase‐conjugated secondary antibodies were used to detect primary antibodies. The ECL detection system (Millipore, Billerica, MA, USA) was used to image protein bands. Primary antibodies included anti‐β‐actin (1:1,000, ab8226, Abcam, Cambridge, MA, USA), anti‐Shh (1:1,000，ab53281，Abcam), anti‐Gli‐1 (1:1,000, ab134906, Abcam), anti‐Ptch1 (1:500, ab53715, Abcam), and anti‐Smo (1:1,000, ab72130, Abcam).
3
Determining Protein Expression Levels
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CSC221, cells treated for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer24 (link). Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), Msi-1 (ab52865, Abcam), Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), Bmi-1 (ab38295; Abcam) were used to detection. α-tubulin (2125, Cell Signaling Technology) and β-Actin (sc-47778; SANTA CRUZ) antibody was used as an internal control. The bands were cut according to the protein size region of interest before intubating with antibodies and then imaged with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Uncropped images of the blot images are presented as additional data (Supple Figs. 10 , 11 ). Bands relative density was calculated based on the density of α-tubulin and actin bands in each sample. Values were demonstrated as arbitrary densitometric units corresponding to signal intensity.
4
Immunohistochemical Characterization of DLBCL
5
Western Blot Analysis of Kidney Proteins
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Proteins were extracted from the kidneys. Equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with anti-SMO (1:10000, ab72130) and anti-GLIS1 (1:1000, ab105873) primary antibodies from Abcam (Abcam Inc., Cambridge, MA). Anti-α-tubulin (1:10000, T5168) from Sigma (Sigma Chemical Co., St. Louis, MO) was used as control. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000, #7074S) from Cell Signaling Technology (Cell Signaling Technology Inc., Danvers, MA) and goat anti-mouse secondary antibody (1:10000, G21040) from Invitrogen (Invitrogen, Carlsbad, CA) were used for detection. Protein bands were visualized using ECL Western blot detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
6
Hedgehog Signaling Pathway Analysis
7
Protein Expression Analysis Protocol
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The total protein was extracted according to the kit. Fifty micrograms of sample were loaded onto sodium dodecyl sulfate–polyacrylamide gels, separated using electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membranes using standard procedures. After blocking with 5% skim milk and washing for 30 min with Tris buffered saline Tween (TBST), the PVDF membrane was immunoblotted with Smo antibody (1:1,000 dilution; ab72130; Abcam), Gli1 antibody (1:1,000 dilution; ab1515796; Abcam), VEGF antibody (1:500 dilution; sc7269; Santa Cruz), and β-actin antibody (1:2,000 dilution; Proteintech, Rosemont, IL, USA) overnight at 4°C. The membranes were then incubated with a horseradish peroxidase–conjugated anti-rabbit or anti-mouse immunoglobulin G secondary antibody.
8
Immunohistochemical Analysis of Ocular Signaling Pathways
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We used a biotin-streptavidin HRP detection system (SP-9000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, People’s Republic of China) to perform immunohistochemistry. Formalin-fixed, paraffin-embedded eye tissue sections (3.5 μm thick) were placed on slides, deparaffinized in xylene, and rehydrated by incubation in graded ethanol baths in PBS. Endogenous peroxidase was blocked with hydrogen peroxide. The sections were then treated with goat serum for 30 min and incubated overnight with the following antibodies: rabbit anti-Smo polyclonal antibody (1:50 dilution; ab72130; Abcam, Cambridge, UK), rabbit anti-Gli1 polyclonal antibody (1:50 dilution; sc-20687; Santa, Santa Cruz, CA, USA), mouse anti-VEGF monoclonal antibody (1:100 dilution; sc-7269; Santa, USA) at 4°C. Subsequently, they were incubated with Biotin-labeled goat anti-rabbit/mouse IgG secondary antibodies (SP-9000; Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, People’s Republic of China) for 15 min at room temperature. The primary antibody was replaced with PBS for the negative controls, and 3,3′-diaminobenzidine was used as the chromogen. The images were observed and captured using an Olympus B201 optical microscope (Olympus, Tokyo, Japan).
9
Smo Expression in Mouse Cardiac CD31+ Cells
10
Smo Expression in Mouse Cardiac CD31+ Cells
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Expression of Smo from mouse cardiac CD31+ cells was evaluated by SDS PAGE using rabbit polyclonal anti-Smo (Abcam, Cat# ab72130) and secondary antibody goat anti-rabbit IgG-horseradish peroxidase (HRP).
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