Anti-Cofilin (phospho S3) 抗体

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (AB12866)

ICC/IF image of ab12866 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab12866, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• Flow Cyt

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Flow Cytometry - Anti-Cofilin (phospho S3) antibody (AB12866)

Flow Cytometry analysis of U-87 MG cells labeling Cofilin (phospho S3) with ab12866. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Anti-Cofilin (phospho S3) antibody (ab12866, red) or with rabbit isotype control (pink) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor^® 488 Goat Anti-Rabbit Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

• ICC/IF

AbReview31575****

Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (AB12866)

Immunocytochemistry/ Immunofluorescence analysis of human neuroblastoma cells labeling Cofilin (phospho S3) with ab12866 at 1/500 dilution. Cells were fixed in paraformaldehyde, permeabilized for 15 minutes in 0.01% Triton X-100, blocked using 5% serum for 30 minutes at 20°C, then incubated with ab12866 at a 1/500 dilution for 2 hours at 20°C. The secondary used was a Dylight 488 conjugated donkey anti-rabbit IgG (H+L) used at a 1/500 dilution.

This image is courtesy of an anonymous abreview.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (AB12866)

Immunofluorescence analysis of Phospho-Cofilin pSer3 was done on 70% confluent log phase PC-3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab12866 at 1 : 250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor^® 488 conjugate at a dilution of 1 : 2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin (1 : 300). Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

• WB

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Western blot - Anti-Cofilin (phospho S3) antibody (AB12866)

All lanes:

Western blot - Anti-Cofilin (phospho S3) antibody (ab12866) at 1 µg/mL

Lane 1:

HeLa whole cell extract at 20 µg

Lane 2:

HeLa treated for overnight with 150 uM of H2O2 whole cell extract at 20 µg

Lane 3:

HeLa treated for overnight with 3 uM of Staurosporine whole cell extract at 20 µg

Lane 4:

COS-7 whole cell extract at 20 µg

Lane 5:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell extract at 20 µg

Lane 6:

MCF7 whole cell extract at 20 µg

Lane 7:

Rat Skeletal Muscle whole cell extract at 20 µg

Lane 8:

A-431 whole cell extract at 20 µg

Lane 9:

A-431 treated for overnight with 150 uM of H2O2 whole cell extract at 20 µg

Lane 10:

Jurkat whole cell extract at 20 µg

Lane 11:

Jurkat treated for overnight with 3 uM of Staurosporine whole cell extract at 20 µg

Secondary

All lanes:

Goat anti-rabbit IgG (H+L), HRP conjugate at 1/2500 dilution

Predicted band size: 18 kDa

Observed band size: 18 kDa

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• WB

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Western blot - Anti-Cofilin (phospho S3) antibody (AB12866)

Peptide Competition and Phosphatase Treatment
Lysates prepared from MDCK cells treated with staurosporine (1) or left untreated (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-5) or treated with lambda phosphatase (6), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with the ab12866 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with : no peptide (1, 2, 6), the non phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4) or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that only the peptide corresponding to cofilin [pS3] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the anti

All lanes:

Western blot - Anti-Cofilin (phospho S3) antibody (ab12866)

Predicted band size: 18 kDa

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• WB

CiteAb

Western blot - Anti-Cofilin (phospho S3) antibody (AB12866)

Cofilin (phospho S3) western blot using anti-Cofilin (phospho S3) antibody ab12866. Publication image and figure legend from Pang, D., Yang, C., et al., 2020, Biol Open, PubMed 31988091.

ab12866 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab12866 please see the product overview.

Suppressed cofilin activity was implicated in PP2-inhibited cellular motility of HepG2 and BEL7402 cells. (A–D) Western blots and quantifications show increased phosphorylation of cofilin-1 in the cells treated with 0, 0.5 and 1.0 μM PP2 for 24 h. (E,F) Wound healing assay and quantifications show stably expressed mutant cofilin-1 (S3A) improves cellular motility of HepG2 cells under the treatment of PP2 (1 μM). (G–J) Western blots and quantifications show decreased protein levels of SSH-1 in the cells treated with 0, 0.5 and 1.0 μM PP2 for 24 h. **p<0.01, ***p<0.001. Scale bar : 200 μm.

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