Proteins were extracted from CRSwNPs tissues or cells using radioimmunoprecipitation assay (Beyotime Institute of Biotechnology), and the concentrations were determined according to the standard protocols of BCA protein assay kit (Beyotime Institute of Biotechnology), respectively. The total protein (30 µg/well) in the supernatant was separated via SDS-PAGE on 10% gel, and then transferred to PVDF membranes. After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with rabbit anti-MMP-2 (1:1,000; no. ab92536; Abcam), rabbit anti-MMP-9 (1:1,000; no. ab76003; Abcam), rabbit anti-TIMP-1 (1:1,000; no. ab211926; Abcam), rabbit anti-integrin β1 (1:1,000; no. ab52971; Abcam), rabbit anti-α-tubulin (1:5,000; no. ab18207; Abcam), rabbit anti-acetyl-α-tubulin (1:1,000; no. ab179484; Abcam) and rabbit anti-β-actin (1:5,000; no. ab8227; Abcam) antibodies. After three washes with TBS with 0.1% Tween-20, the immunoblots were incubated for 1 h at room temperature with goat alkaline phosphatase-labeled anti-rabbit antibody (1:1,000; cat. no. 14708; Cell Signaling Technology, Inc.). The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology). The blots were semi-quantified using ImageJ software (version 1.47; National Institutes of Health).
Ab179484
Manufactured by Abcam
Sourced in United Kingdom
Ab179484 is a laboratory equipment product offered by Abcam. It serves as a core function for research purposes. The detailed specifications and intended use of this product are not available for an unbiased and factual presentation at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab7291, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Ab18207, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ab76003, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Ab52971, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab211926, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab184601, by Abcam (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Sangon (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Ecl kit, by Advansta (1 mentions)
Nitrocellulose membrane, by Promega (1 mentions)
Ma5 15738, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
14 protocols using ab179484
1
Protein Expression Analysis in CRSwNPs
2
Western Blot Analysis of Protein Extracts
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Whole-cell protein extracts were prepared using RIPA lysis buffer (PC101, EpiZyme, China). Proteins were separated on a 10% SDS/PAGE gel and electroblotted on to an Immobilon-P membrane (Millipore). The membrane was blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline containing Tween 20 (TBST) buffer (20 mM Tris/HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20), then incubated with the primary antibody overnight at 4°C, washed with TBST buffer, and incubated with HRP-conjugated secondary antibody for 1.5 h at room temperature. After being washed with TBST buffer, blots were developed with SuperSignal West Pico or Femto chemiluminescent substrate (Pierce) or ECL Plus Western blotting detection reagents (GE Healthcare). Antibodies used were rabbit monoclonal to α-tubulin (ab179484, Abcam, Cambridge, U.K.), rabbit monoclonal to EGFP (ab184601, Abcam, Cambridge, U.K.). Proteins recognized by the antibodies were detected by ImageQuant LAS 500 (GE, CT, U.S.A.) using HRP-conjugated Goat Anti-Rabbit secondary antibody (BBI, Sangon Biotech, China).
3
Western Blot for Protein Analysis
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The tissues extracted from motor cortices and ischemic penumbra areas at different time points were lysed in precooled RAPI buffer. Total proteins were separated by 10% SDS‐PAGE gels; then, the protein in gels was transferred onto PVDF membranes. After blockage in 3% bovine serum albumin for 2 h, the membranes were incubated with the primary antibodies: (1) mouse anti‐tubulin (1:1000, ab7291; Abcam); (2) rabbit anti‐α‐Ac‐Tub (1:1000, ab179484; Abcam); (3) rabbit anti‐MEC17 (1:1000, ab184778; Abcam) overnight at 4°C; and subsequently with the secondary antibodies used were: HRP‐conjugated goat anti‐rabbit or rabbit anti‐mouse ones 1 hour at room temperature. We used the ECL Kits (Advansta) to develop membranes using imaging system (Bio‐Rad). The relative intensities of the bands were analyzed using NIH Image J software.
4
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from cells with RIPA lysis buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Sigma-Aldrich), and 20–30 μg of proteins were size-separated using NuPage BisTris gels 4–12% (Invitrogen) and transferred to nitrocellulose membranes (Promega). Membranes were blocked with PBS containing 0.1% Tween-20 and 5% non-fat dry milk and incubated with antibodies to CCDC141 (1:1000 dilution, SAB3500670, Sigma-Aldrich), Ac-tubulin (1:1000 dilution, ab179484, Abcam), Proliferating Cell Nuclear Antigen (PCNA 1:2000 dilution, 2586, Cell Signaling Technologies) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH 1:10,000 dilution, G9545, Sigma-Aldrich and MA5-15738, Thermofisher). Anti-rabbit and mouse IRDye680 or IRDye800 (Licor) were used as secondary antibodies (1:10,000 dilution). Immunoblots were scanned with the Odyssey® Fc Imaging System (Licor).
5
Immunofluorescence Analysis of CCDC141 Variants
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HEK293T cells were seeded on cover slips and transiently transfected with either WT or CCDC141 variants using Lipofectamine, and after 48 h were utilized for immunofluorescence. HEK293 cells stably expressing either WT or CCDC141 variants were used for immunofluorescence 24 h after seeding. Cells were fixed in 4% paraformaldehyde for 15 min, then were permeabilised with 0.1% Triton X-100 in PBS for 30 min and blocked with blocking buffer [10% bovine serum albumin (BSA) in PBS] for 30 min. Anti-FLAG (dilution 1:1000; F1804, Sigma-Aldrich), anti-Ac-tubulin (dilution 1:500; ab179484, Abcam), and anti-pericentrin (dilution 1:2000; ab4448, Abcam) diluted in 3% BSA/PBS were used to incubate cells for 2 h at RT. Secondary Ab, anti-Mouse IgG-FITC (Invitrogen), at a 1:1000 dilution in 3% BSA/PBS was applied for incubation for 45 m at RT. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) by using Vectorshield mounting medium with DAPI (Vector laboratories). Images were taken by a LSM 510 laser scanning confocal microscope (Zeiss) and processed using Adobe Photoshop CS6.
6
Dental Pulp Stem Cell Encapsulation
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HeP-bFGF was
gradually added to the complete cell culture medium
(α-MEM supplemented with 15% FBS and 1% S/P) in an ice bath
for 12 h. Then, the solution was filtered by a bottle-top filter (0.22
μm pore size, Biofil). Next, DPSCs (1 × 106 cells/mL)
were mixed in hydrogels in an ice bath. After culturing, the culture
plate was put in a 37 °C and 5% CO2 incubator for
20 min till gel formation occurred. A total of 180 μL/well medium
was added to prevent the gel from dehydrating. Light microscopy was
used to affirm the homogeneous cell encapsulation. The regular medium
was changed every 3 days. The stem cells were confirmed by immunofluorescence
staining with α-tubulin (ab179484, Abcam).
gradually added to the complete cell culture medium
(α-MEM supplemented with 15% FBS and 1% S/P) in an ice bath
for 12 h. Then, the solution was filtered by a bottle-top filter (0.22
μm pore size, Biofil). Next, DPSCs (1 × 106 cells/mL)
were mixed in hydrogels in an ice bath. After culturing, the culture
plate was put in a 37 °C and 5% CO2 incubator for
20 min till gel formation occurred. A total of 180 μL/well medium
was added to prevent the gel from dehydrating. Light microscopy was
used to affirm the homogeneous cell encapsulation. The regular medium
was changed every 3 days. The stem cells were confirmed by immunofluorescence
staining with α-tubulin (ab179484, Abcam).
7
Immunofluorescence Staining of Oocytes
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The procedure of Immunofluorescence was carried out as previously described (5 (link)). Briefly, the oocytes, fixed with 4% paraformaldehyde (Byotime), were punched with 0.5% Triton X-100 (Byotime) for 10 min, and then globally blocked in QuickBlock™ Blocking Buffer for Immunol Staining (Byotime) for 1 h. The oocytes were cultivated for 8 h at 4°C in first antibodies diluted in QuickBlock™ Primary Antibody Dilution Buffer (P0262, Byotime). After washed thoroughly in PBS, the samples were stained with the diluted Alexa Fluor 488 or 555 goat anti-rabbit or anti-mouse IgG (H + L) (1:600, Invitrogen, Carlsbad, CA, United States) for 2 h at room temperature, and then stained with DAPI (C1006, Beyotime) for 3 min. The antibodies are listed as follows: rabbit anti-SIRT1 (ab189494, 1:100, Abcam), mouse anti-α-tubulin (ab7291, 1:600, Abcam), rabbit anti-α-tubulin (acetyl K40) (ab179484, 1:500, Abcam), rabbit anti- Anti-p53 (acetyl K382) (ab75754, 1:200, Abcam)rabbit anti-GRP78 (ab108615, 1:100, Abcam), and rabbit anti-GOLPH3 (AP6024, 1:100, Bioworlde), rabbit anti-LAMP1 (ab208943, 1:100, Abcam). Finally, the signals were examined with a Zeiss Axio Observer D1 microscope (Carl Zeiss, Inc., Thornwood, NY). The procedure of staining and settings of microscope were the same for each group.
8
Western Blotting of Stress Proteins
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Cells were treated as indicated and Western blotted as previously described (Liu et al., 2015 (link)). Briefly, the cells were lysed and assessed using 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SurePAGE, Bis-Tris gels, Genscript, Nanjing, China). Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States) and probed with primary antibodies against HSPA6 (sc-374589, Santa Cruz Biotechnology), HSPA1A (PA534772, Invitrogen), HSPA8 (PA5-27337, Invitrogen), HSPB1 (PA1017, Invitrogen), EV71 VP1 (GTX132339, GeneTex), CVA6 VP1 (GTX132346, GeneTex), GAPDH (ab9485, Abcam) or α-Tubulin (ab179484, Abcam). Protein bands were visualized with Pierce™ ECL Western blotting substrate (Thermo Scientific, Rockford, IL, United States). Protein levels were determined by scanning the bands’ intensities and analyzed using Quantity One software (Bio-Rad, Hercules, CA, United States).
9
Immunofluorescence Staining of Neuronal Structures
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For immunofluorescence staining, sections of the brain or primary cultured neuronal dishes were fixed using paraformaldehyde. Subsequently, a blocking solution was prepared with 10% normal goat serum and 0.5% Trition X‐100. The samples were then incubated in the blocking solution for 2 h at room temperature prior to the staining procedure. Furthermore, respective primary antibodies [rabbit anti‐α‐Ac‐Tub (ab179484, 1:1000, Abcam) and mouse anti‐PSD95 (ab2723, 1:1000, Abcam)] were added to the samples and incubated overnight at 4°C. Subsequently, the sections or dishes were incubated with secondary antibodies [Alexa Fluor 488‐conjugated goat anti‐mouse and Alexa Fluor 555‐conjugated goat anti‐rabbit (Invitrogen)] at room temperature for 2 h. Confocal microscopy was performed to capture images (Carl Zeiss 880), which were then processed using Zen 2011 software (Carl Zeiss). Measurements were taken in a blinded manner. An average of data from five mice or dishes obtained using Image J (National Institutes of Health) was recorded.
10
Tubulin Acetylation Analysis in HEK293T Cells
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HEK293T cells were
treated for 24 h with 10 or 30 μM25 or 10 μM
Tubacin or DMSO control. Cell lysates were analyzed by Western blot
using the NuPAGE electrophoresis and transfer system (Invitrogen),
and proteins detected with primary antibodies anti-α tubulin-K40ac
(1:2000, Abcam, ab179484) and anti-α tubulin (1:2000, Abcam,
ab7291) and secondary antibodies IRDye 800CW (1:5000, ThermoFisher,
A32735) and IRDye 488CW (1:5000, ThermoFisher, A11029). Immunoblots
were imaged on a Li-Cor Odyssey CLx.
treated for 24 h with 10 or 30 μM
Tubacin or DMSO control. Cell lysates were analyzed by Western blot
using the NuPAGE electrophoresis and transfer system (Invitrogen),
and proteins detected with primary antibodies anti-α tubulin-K40ac
(1:2000, Abcam, ab179484) and anti-α tubulin (1:2000, Abcam,
ab7291) and secondary antibodies IRDye 800CW (1:5000, ThermoFisher,
A32735) and IRDye 488CW (1:5000, ThermoFisher, A11029). Immunoblots
were imaged on a Li-Cor Odyssey CLx.
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