Cells were seeded and treated as above, except fresh aspirin-containing medium was added 1 h before harvesting, as previously described [20 (link)]. Whole cell lysates were harvested on days 1 and 4 with and without aspirin in Laemmli Buffer (Bio Rad, Hercules, CA) and boiled for 10 min at 100 °C. Lysates were resolved using SDS-PAGE using 4–12 % bis–tris NuPAGE gels in MES running buffer (Invitrogen, Grand Island, NY) following the manufacturer’s protocol. The proteins were transferred using Invitrogen Xcell II blotting apparatus to a PVDF membrane (Invitrogen, Grand Island, NY). Following transfer, the membranes were blocked in 5 % w/v milk in tris(hydroxymethyl)aminomethane (TRIS)-buffered saline supplemented with 0.1 % tween-20 (Sigma, Saint Louis, MO) for 1 h. Membranes were probed with either phosphorylated GSK3β (Ser 9; 9336), total GSK3β (9315), phosphorylated Src family (Tyr 416; 2101), total Src (ab47405, Abcam, Cambridge, MA), ACTB (β-actin) (4967), and TUBB (β-tubulin) (2146) primary antibody followed by incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (7074). Protein bands were visualized using enhanced chemiluminescent reagent (Perkin-Elmer, Waltham, MA). All antibodies were purchased from Cell Signaling Technology (Beverly, MA) unless otherwise noted. Densitometry was performed using Image J analysis software (NIH).
Ab47405
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab47405 is a lab equipment product offered by Abcam. It is a core functional item used in scientific research and applications. The detailed specifications and intended applications of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Ab39688, by Abcam (2 mentions)
Ab155549, by Abcam (2 mentions)
Ab199396, by Abcam (2 mentions)
Ab131442, by Abcam (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Santa Cruz Biotechnology (2 mentions)
β actin, by Abcam (2 mentions)
Ab51067, by Abcam (2 mentions)
Ab188224, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab18199, by Abcam (2 mentions)
Zen 2009, by Zeiss (2 mentions)
Goat anti rabbit igg conjugated with alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Multiphoton microscope lsm t pmt system, by Zeiss (2 mentions)
Goat anti mouse igg conjugated with alexafluor 488, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescent reagent, by PerkinElmer (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
11 protocols using ab47405
1
Western Blot Analysis of Aspirin Effect
2
Tissue Microarray Immunohistochemistry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A tissue microarray (TMA) was constructed with the Tissue Microarrayer (Beecher Instruments, Silver Springs, USA). Each sample was represented in quadruplicates. IHC was carried out using rabbit polyclonal anti-SRC (Abcam: ab47405, Cambridge, UK) (dilution 1∶200). Positive breast tumor tissue and two negative controls were assessed by IHC. The negative controls were created via omission of the primary antibody and incubation of the slides in PBS, followed by replacement of the primary antibody with normal rabbit serum. The final scores (median of the four scores) were obtained according to staining intensity of the cytoplasm or membrane and were described as negative/weak (score 0–1) or positive (score 2–3). The samples were scored blind with respect to clinical data of the patient.
3
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was rst extracted using SDS-PAGE and then transferred to a PVDF membrane (Thermo, USA). Afterwards, the PVDF membrane was incubated with 1:1000 dilution of antibody: TRIM47 (ab155549, Abcam, USA), BAP1 (ab199396, Abcam, USA), P21 (ab188224, Abcam, USA), P53 (ab131442, Abcam, USA), SRC (ab47405, Abcam, USA), MET (ab51067, Abcam, USA), and c-Myc (ab39688,Abcam, USA). The membrane was washed and incubated with a 1:2000 dilution of horseradish peroxidaseconjugated goat anti-rabbit (Santa Cruz, USA). SuperSignalMT West Puico PLUS (Termo Scienti c, USA) was used to develop the blot, using b-actin (ab8226, Abcam, USA) as the loading control. All experiments were performed in triplicate.
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was first extracted using SDS-PAGE and then transferred to a PVDF membrane (Thermo, USA). Afterwards, the PVDF membrane was incubated with 1:1000 dilution of antibody: TRIM47 (ab155549, Abcam, USA), BAP1 (ab199396, Abcam, USA), P21 (ab188224, Abcam, USA), P53 (ab131442, Abcam, USA), SRC (ab47405, Abcam, USA), MET (ab51067, Abcam, USA), and c-Myc (ab39688,Abcam, USA). The membrane was washed and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-rabbit (Santa Cruz, USA). SuperSignalMT West Puico PLUS (Termo Scientific, USA) was used to develop the blot, using b-actin (ab8226, Abcam, USA) as the loading control. All experiments were performed in triplicate.
5
Immunofluorescence Analysis of Cytoskeletal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells cultured on coverslips
were fixed with cold methanol for 10 min, blocked in PBS containing
10% goat serum and 0.01% Triton X-100, and then stained with antibodies
against α-tubulin (T5168, Sigma), acetylated tubulin (T6793,
Sigma), MAP1B (4528, Sigma), or c-Src (ab47405, Abcam). Bound primary
antibodies were detected using an AlexaFluor 594-conjugated goat anti-mouse
IgG secondary antibody (Invitrogen). Coverslips were mounted with
ProLong Gold Antifade reagent (Invitrogen) for fluorescence microscopy.
Slides were examined by an Olympus BH2-RFCA fluorescence microscope
with a 60× objective equipped with a Sony DXC-950 3CCD color
camera and Northern Eclipse version 5.0 from Empix Imaging (Mississauga,
ON).
were fixed with cold methanol for 10 min, blocked in PBS containing
10% goat serum and 0.01% Triton X-100, and then stained with antibodies
against α-tubulin (T5168, Sigma), acetylated tubulin (T6793,
Sigma), MAP1B (4528, Sigma), or c-Src (ab47405, Abcam). Bound primary
antibodies were detected using an AlexaFluor 594-conjugated goat anti-mouse
IgG secondary antibody (Invitrogen). Coverslips were mounted with
ProLong Gold Antifade reagent (Invitrogen) for fluorescence microscopy.
Slides were examined by an Olympus BH2-RFCA fluorescence microscope
with a 60× objective equipped with a Sony DXC-950 3CCD color
camera and Northern Eclipse version 5.0 from Empix Imaging (Mississauga,
ON).
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from the tissues using RIPA lysis buffer as previously described [44 (link)]. Cell lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4 °C, which included an anti-phospho Src antibody (ab185617, Abcam, Cambridge, UK, 1:1000 dilution), anti-Src antibody (ab47405, Abcam, 1:600 dilution), anti-TGF-β1 antibody (sc-146, Santa Cruz Biotechnology, Dallas, TX, USA, 1:400 dilution), anti-fibrinogen antibody (sc-18029, Santa Cruz Biotechnology, 1:800 dilution), anti-α-SMA antibody (sc-53015, Santa Cruz Biotechnology, 1:800 dilution), and anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, 1:1000 dilution). The membranes were washed and incubated with the secondary antibodies for 2 h at room temperature. Protein expression was detected using a chemiluminescence system (Millipore, Billerica, MA, USA) according to the manufacturer’s specifications.
7
Breast Cancer Cell Culture and Antibody Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 human breast cancer cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in ATCC-formulated Leibovitz's L-15 Medium (Catalog No. 30-2008). Cells were supplemented with heat inactivated fetal bovine serum to a final concentration of 10%, and incubated at 37°C in a free gas exchange with atmospheric air. Mouse monoclonal anti-FAK (ab72140), rabbit polyclonal anti-Src(ab47405), -paxillin(ab39537), and -β-actin antibodies (ab8227), and goat anti-mouse (ab1500117), goat anti-rabbit IgG (ab150077) antibodies were all obtained from Abcam Corporation.
8
Protein Expression Analysis in Mouse Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse tissues and cells for protein lysates were lysed in RIPA buffer, homogenized by a Tissue LyserII (Qiagen, Venlo, Netherlands) and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were subjected to 8% or 10% SDS-PAGE and transferred to a PVDF membrane. Protein expression was analyzed using rabbit anti-ASAP1 (ab12423, 1:2500, Abcam, Cambridge, UK), rabbit anti-FAK (06–543, 1:1000, Merck Milipore, Darmstadt, Germany), rabbit anti-phospho FAK Y397 (44-624G, 1:1000, Thermofisher), rabbit anti-Src (ab47405, 1:1000, abcam), rabbit anti-phospho Src Y416 (#6943, 1:1000, Cell Signaling), rabbit anti-phospho AKT T308 (#9275, 1:1000, Cell Signaling), rabbit anti-AKT (sc-8312, 1, 1:200, Santa Cruz), rabbit anti-ERK (sc-93, 1:200, Santa Cruz), mouse anti-phospho ERK (#9106, 1:1000, Cell Signaling), rabbit anti-p38 (sc535, 1:200, Santa Cruz), rabbit anti-phospho p38 (#4511, 1:1000, Cell Signaling). Probing the membranes with mouse anti-vinculin antibodies (V4139, 1:5000, Sigma) served as a loading control.
9
Visualizing Lipid Raft Dynamics in Cells
10
Immunofluorescence Microscopy of L1, Caveolin-1, and Src
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2A2-L1s cells were plated in T75 flasks in DMEM supplemented with 10% bovine serum (BS) and grown to 75 to 85% confluence (Dou et al., 2011 (link)). Cells were treated with 25 mM EtOH, 5 μm filipin, or both for 1 hour. Cells were harvested in phosphate buffered saline (PBS) plus 2 mm EDTA, fixed in 4% paraformaldehyde for 30 minutes, blocked with PBS supplemented with 5% BS, and incubated with L1 mAb 5G3 (Dou et al., 2011 (link)), caveolin-1 polyclonal antibody (AB18199; Abcam), or Src polyclonal antibody (Ab47405; Abcam, Cambridge, MA) in PBS/BS at room temperature for 2 hours. Cells were washed 3 times with PBS and incubated with goat anti-mouse IgG conjugated with Alexa Fluor-488 and goat anti-rabbit IgG conjugated with Alexa Fluor 546 (Invitrogen, Grand Island, NY) in PBS/BS. Cells were washed again with PBS and fixed in paraformaldehyde. Images were captured using a Zeiss Multiphoton microscope LSM T-PMT system and Zen 2009 software from Carl Zeiss (Carl Zeiss International, Jena, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!