Cells or tissue sections were fixed in 4% paraformaldehyde. After washing 3 times with PBS for 3 min each time, the samples were incubated in 10% normal donkey serum in PBS for 20 min, and then incubated with MSI2 (Abcam, ab76148), CD44v6 (Abcam, ab78960) or Notch1 (Abcam, ab52627) primary antibodies in PBS at 4 °C for overnight. Incubated the samples with fluorochrome-conjugated secondary antibodies (1:400, Alexa Fluor®488 donkey anti-rabbit lgG, or Alexa Fluor®594 donkey anti-rabbit lgG, or Alexa Fluor®594 donkey anti-mouse lgG, life technologies) for 30 min, and subsequently incubated with DAPI. The images were observed and collected under fluorescence microscope. Three random fields in 200× were selected for quantification. ImageJ software was used to analyze the fluorescence intensity of CD44v6, MSI2 and Notch1. The experiments were repeated independently three times.
Ab76148
Manufactured by Abcam
Sourced in United Kingdom
Ab76148 is a recombinant protein that serves as a control for the detection of proteins by immunoassay techniques. The product is intended for laboratory research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ab140631, by Abcam (3 mentions)
Ab52865, by Abcam (3 mentions)
Ab78960, by Abcam (2 mentions)
Sc 9864, by Santa Cruz Biotechnology (2 mentions)
Ab32503, by Abcam (2 mentions)
Alexa fluor 488 donkey anti rabbit lgg, by Thermo Fisher Scientific (1 mentions)
Ab52627, by Abcam (1 mentions)
4 oh tamoxifen, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab8978, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Protease inhibitor tablet, by Merck Group (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
A3854, by Merck Group (1 mentions)
Ab31013, by Abcam (1 mentions)
Fluorescein isothiocyanate (fitc), by Abcam (1 mentions)
Ab81299, by Abcam (1 mentions)
21 protocols using ab76148
1
Immunofluorescence Analysis of Cancer Markers
2
Evaluating Msi2-Dependent Factors in Leukemic Stem Cells
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To check the expression of Hoxa9, Ikzf2, and Myb in LSCs, c-Kithi (top 10–12%) bone marrow cells (LSCs) from Msi2 f/f Cre-ER- and Msi2 f/f Cre-ER+ mice were sorted and were left untreated or treated with 600 nM 4-OH Tamoxifen (Sigma-Aldrich) for 68 h in BMT medium. One hundred thousand cells were collected, washed once with PBS, and then lysed in 1× Laemmli sample buffer (BioRad). LSCs were also sorted from quaternary MLL-AF9 DsRed leukemia mice, then were transduced with lentiviral shRNAs against murine Msi2 (sh331 and sh332) or shRNA against Luciferase. Transduced cells were selected with 2 μg/mL puromycin. After 72 h of transduction, cells were collected, washed in PBS and lysed in 1× Laemmli sample buffer. For analysis in LSKs, one hundred thousand LSK cells from 3 week pIpC treated Msi2 f/f Cre- and Msi2 f/f Cre+ mice were sorted, washed with PBS and lysed in 1× Laemmli sample buffer. Cell lysate was run on 4–15% SDS-PAGE gels, transferred onto nitrocellulose membrane and then probed with antibodies against MSI2 (Abcam, ab76148, dilution 1:1000), HOXA9 (Abcam, ab140631; dilution 1:1000), IKZF2 (Santa Cruz, sc-9864, dilution 1:1000), MYB (Millipore, 05-175, dilution 1:1000), and ACTB (beta-actin-HRP, dilution 1:30,000) (Sigma-Aldrich, A3854).
3
Western Blot Analysis of EMT Markers
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Tissues and cells were lysed in cell lysis buffer supplemented with protease inhibitor cocktail on ice for 30 minutes. Then, the samples were centrifuged at 12,000 rpm for 15 minutes, and the supernatants containing the proteins were collected. The proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with the primary antibodies at 4°C overnight. The next day, after the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour, the bands were captured and visualized with a chemiluminescence imaging system (Shanghai, China). The primary antibodies used were as follows: anti-MSI2 (ab76148, Abcam); anti-E-cadherin (ab1416, Abcam); anti-vimentin (ab8978, Abcam); and anti-N-cadherin (ab76011, Abcam).
4
IP-Western Blot Characterization of RNA-binding Proteins
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K562 cells were collected by spinning down at 1,500 rpm for five minutes at 4°C, washed twice with PBS, and then resuspended thoroughly at 2×107 (link) per ml in 1X Ripa buffer (BP-115- Boston BioProducts) with freshly added DTT (1mM) and proteinase inhibitor cocktail. The cells were incubated for 30 min on ice. Supernatant was then collected after the mix was spun at 14,000 rpm for 30 min at 4°C. For each immunoprecipitation assay, 250 ul of cell extract was mixed with 750 ul of 1X Ripa buffer 2 ug of anti-mouse/anti-SYNCRIP antibody or 2 ug anti-rabbit/anti-MSI2 and 50 ul agarose beads. For RNA independent assay, lysates were treated with RNase A (1ug/ml) for 30 min at 37°C prior to coimmunoprecipitation reactions. After rotating at 4°C overnight, beads were washed 5 times with 1X Ripa buffer and boiled with 1X Lamine protein running buffer.
For immunoblot analysis, cells were counted and washed twice with cold PBS prior to collection. ~ 250,000 were resuspended and lysed in 40 ul 1X Lamine protein running buffer and boild for 5 minutes. Whole cell lysates were run on 4%–15% gradient SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blotted for SYNCRIP (MAB11004 or 05-1517-Milipore), IKZF2 (sc-9864; Santa Cruz), HOXA9 (07–178; Millipore and ab140631, Abcam), MYC (5605S; Cell Signaling), MSI2 (ab76148; Abcam), and ACTIN (A3854; Sigma-Aldrich).
For immunoblot analysis, cells were counted and washed twice with cold PBS prior to collection. ~ 250,000 were resuspended and lysed in 40 ul 1X Lamine protein running buffer and boild for 5 minutes. Whole cell lysates were run on 4%–15% gradient SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blotted for SYNCRIP (MAB11004 or 05-1517-Milipore), IKZF2 (sc-9864; Santa Cruz), HOXA9 (07–178; Millipore and ab140631, Abcam), MYC (5605S; Cell Signaling), MSI2 (ab76148; Abcam), and ACTIN (A3854; Sigma-Aldrich).
5
Immunoblot Analysis of TGFβ Signaling
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For immunoblot analysis, Ro treated and DMSO control MOLM13 or K562 cells (routinely at 0.5 × 106 cells mL−1) were counted and washed twice with cold PBS before collection. 1–5 × 106 cells were resuspended and lysed in 250 μl of 1× RIPA Buffer supplemented with Protease Inhibitor Tablets (Sigma-Aldrich) buffer for 30 min on ice. After centrifugation at 20,820 × g on a top-bench centrifuge, lysate (supernatant) was collected and total protein quantified by BCA (Thermo Scientific). Cell lysates were separated by 4–15% SDS–PAGE and transferred to 0.45 μm nitrocellulose membrane. Membranes were blocked and were blotted overnight (4 °C) for TGβR1 (ab31013, ABCAm, 1:750 dilution), SMAD3 (9523S, Cell Signaling Technology, 1:750 dilution), HOXA9 (07–178, Millipore, for drug dose-dependent experiments and ab140631, ABCAm; 1:1000 dilution for time-course experiments), c-MYC (5605, Cell Signaling Technology; 1:1,000 dilution), P21 (2947 S, Cell Signaling Technology, 1:750 dilution), MSI2 (ab76148, ABCAm; 1:2,000 dilution) and β-ACTIN-HRP conjugated (A3854, Sigma-Aldrich; 1:20,000 dilution) and developed by Hyperfilm ECL (GE Healthcare) with ECL and pico-ECL reagents (Thermo Scientific). Uncropped and unprocessed scans are included in the Source Data file.
6
Immunofluorescence Analysis of DNA Damage and RNA-Binding Proteins
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Cells were cultured on 22 × 22 mm2 cover slips in six‐well plates, then subjected to irradiation and fixed with 4% paraformaldehyde for 30 min at specified time points. Subsequently, the cells were permeabilized using a 0.5% Triton X‐100 buffer, followed by blocking with 1% BSA (bovine serum albumin) for 1 h at room temperature. The cells were then incubated overnight at 4°C with primary antibodies against γ‐H2AX (Ser139) (Abcam, ab81299), MSI2 (Abcam, ab76148), and RBM17 (Abcam, ab204333). After washing twice with PBS, the cells were treated with FITC (fluorescein isothiocyanate)‐labeled anti‐mouse antibody (Abcam) or Texas Red‐labeled anti‐rabbit antibody (Abcam) at room temperature for 2 h. Nuclei were stained with DAPI (4,6‐diamino‐2‐phenyl indole) for 15 min in the dark. Confocal microscopy (Zesis 880) with the NIS‐Elements Viewer 4.20 capture system was used to capture images. Six slices of each specimen were observed, and five high‐power visual fields were randomly selected. Protein co‐localization analysis was conducted using ImageJ software, analyzing images of MSI2 and RBM17 in 100 nuclei from three independent experiments. The Pearson correlation coefficient and overlap coefficient were used as statistical measures to quantify co‐localization, with measurements performed using the ImageJ plugin Colocalization Finder.
7
IP-Western Blot Characterization of RNA-binding Proteins
8
Immunohistochemical Analysis of CD44v6 and MSI2
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IHC staining with antibodies against CD44v6 (Abcam, ab78960), MSI2 (Abcam, ab76148) was performed to detect protein expression levels. The intensity of staining was scored on a scale as negative (0, no staining), weak (1, light yellow), moderate (2, brown), or strong (3, brown red). The extent of the staining was evaluated according to the percentage of positive areas of cells in relation to the whole area, was scored on a scale of 0–4, 0 (0), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Protein expression levels (range 0–12) were calculated by multiplying the staining intensity and positive staining score. Then, we divided the patients into two groups (grade<6, low expression; grade ≥ 6, high expression) and performed survival analysis. Assessment of IHC staining scores was independently performed by two pathologists (Dr. Yaqi Duan and Dr. Xi Wang, Department of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology), who were blinded to the clinical data. Examples of the staining intensity grades were shown in Additional file 1 : Figure S1B-S1D.
9
Immunoprecipitation of GFP-tagged Proteins
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HEK-293-Flp-In T-Rex cells tagged with EGFP, EGFP-MAB21L1 or mutant EGFP-MAB21L1 were seeded in T-25 flask in culturing media supplemented with 1 μg/ml tetracycline. Cells were harvested by trypsin-EDTA, washed by PBS after 12 hrs of tetracycline treatment and lysed with Nonidet P-40 lysis buffer (50mM Tris, pH 8.0, 150mM NaCl, 1.0% Nonidet P-40) in the presence of protease inhibitor(Roche Applied Science) For each immunoprecipitation, 400 μl of cell lysate were incubated with anti-TBL1XR1 antibody (ab24550,Abcam) and anti-MSI2 antibody(ab76148,Abcam) for 5 h at 4°C. Then 20 μl of Dynabeads protein A (Thermo Fischer) were added and rotated for 2 h at 4°C. Bound immune complexes were washed three times with phosphate-buffered saline. For immunoprecipitation of GFP-tagged proteins, Cell lysis and GFP pulldown was perform using GFP Tag Immunomagnetic Beads (Sino Biologicals) according to manufacturer instructions. The immune-complexes were analysed by Western blotting.
10
Immunohistochemical and Immunofluorescence Analysis of Prostate Cancer Tissue Microarray
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The prostate cancer tissue microarray contained 61 prostate cancer tissue spots and 39 para cancer spots of 64 patients (OUTDO, Shanghai, China). immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described previously.33 Briefly, IHC was performed on the paraffin embedded tissue microarray using anti–MSI2 (1:200). IHC images were captured by a Leica DM5500 B microscope. Protein expression levels were scored by multiplying the percentage of positive cells and immunostaining intensity. The percentage was scored as follows: nonpositive cells as 0 points, 1%‐25% as 1 point, 26%‐50% as 2 points, 51%‐75% as 3 points and 76%‐100% as 4 points. The staining intensity was scored as follows: no positive staining as 0 points, weak staining as 1 point, moderate staining as 2 points and strong staining as 3 points. The final scores were obtained according to above terms: 0‐3 was weak expression, 4‐8 was moderate expression, and 9‐12 was high expression.
Immunofluorescence was performed using anti–human MSI2 (2:100, ab76148, Abcam) and anti–human AR (1:200, NBP2‐44789, Novus). IF images of tissues were acquired using a Leica DMi8 fluorescence microscope. Correlation rates were calculated using ImageJ software.
Immunofluorescence was performed using anti–human MSI2 (2:100, ab76148, Abcam) and anti–human AR (1:200, NBP2‐44789, Novus). IF images of tissues were acquired using a Leica DMi8 fluorescence microscope. Correlation rates were calculated using ImageJ software.
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