- MA5-11705 - Invitrogen Antibodies FLNA antibody

Submitted references The Unfolded Protein Response Sensor PERK Mediates Stiffness-Dependent Adaptation in Glioblastoma Cells.

Khoonkari M, Liang D, Lima MT, van der Land T, Liang Y, Sun J, Dolga A, Kamperman M, van Rijn P, Kruyt FAE
International journal of molecular sciences 2022 Jun 10;23(12)

Inducible microRNA-200c decreases motility of breast cancer cells and reduces filamin A.

Ljepoja B, Schreiber C, Gegenfurtner FA, García-Roman J, Köhler B, Zahler S, Rädler JO, Wagner E, Roidl A
PloS one 2019;14(11):e0224314

Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actin.

Cukier IH, Li Y, Lee JM
FEBS letters 2007 Apr 17;581(8):1661-72

CD28 interaction with filamin-A controls lipid raft accumulation at the T-cell immunological synapse.

Tavano R, Contento RL, Baranda SJ, Soligo M, Tuosto L, Manes S, Viola A
Nature cell biology 2006 Nov;8(11):1270-6

Calpain 3 is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components.

Taveau M, Bourg N, Sillon G, Roudaut C, Bartoli M, Richard I
Molecular and cellular biology 2003 Dec;23(24):9127-35

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of Filamin using Filamin Monoclonal Antibody (Product # MA5-11705) on NIH3T3 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Filamin was performed by loading 7.5 µg of HeLa cell lysate per well onto an SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% milk for 1 hour at room temperature. The membrane was probed with a Filamin monoclonal antibody (Product # MA5-11705) at a dilution of 1:200 overnight at 4C, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:10,000 for 1 hour at room temperature. Detection was performed using chemiluminescent substrate. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Filamin A Monoclonal Antibody (FLMN01 (PM6/317)) (Product # MA5-11705) and a 180kDa band corresponding to Filamin A was observed across all cell lines tested except LNCaP and HEK-293 which are known to have low expression of Filamin A (DOI: 10.18632/oncotarget.9472). Whole cell extracts (50 µg lysate) of DU 145 (Lane 1), LNCaP (Lane 2), HeLa (Lane 3), HEK-293 (Lane 4) and A549 (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1µg/mL dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Filamin Antibody was performed using 70% confluent log phase Ramos 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 Filamin A (FLMN01 (PM6/317)) Mouse Monoclonal Antibody (Product # MA5-11705) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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 (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Filamin A was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Filamin A Monoclonal Antibody (FLMN01 (PM6/317)) (Product # MA5-11705) at 2µg/mL dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing predominantly cytoplasmic localization. Panel e represents HEK-293 cells showing lower expression of Filamin A. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Formalin-fixed, paraffin-embedded human skin stained with Filamin antibody using peroxidase-conjugate and AEC chromogen. Note cytoplasmic staining of smooth muscle cells surrounding blood vessels.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 PERK-deficient GG16 cells are impaired in cellular adaptation to increasing stiffness which is linked with aberrant FLNA expression. ( A , E ) GG16-WT and PERK-KO cells were cultured for 6 days on hydrogels with different stiffnesses and stained with Dapi TM (Blue), Alexa fluor TM 546-Phalloidin (Red), and FLNA--Alexa fluor TM 488 (Green). Cell morphology, F-Actin, and FLNA expression was quantified and is depicted in ( B - D ) and ( F - H ) for GG16-WT and PERK-KO cells, respectively. Cell morphology (from round to elongated), F-Actin, and FLNA expression altered gradually in a stiffness-dependent manner in GG16-WT cells, which was not seen in PERK-deficient cells. * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Inhibition of F-Actin polymerization mimics phenotype of PERK-deficient cells by impairing cellular adaption to matrix stiffness. ( A , E ) GG16-WT cells treated with latrunculin B were cultured for 6 days on hydrogels of varying stiffness. Cells were stained for F-Actin, FLNA, and PERK together with the nuclei and imaged with confocal microscopy. Specific fluorescence was quantified for FLNA ( B ), F-Actin ( C ), cell elongation ( D ), and PERK expression ( F ). ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Inhibition of PERK kinase activity partially impairs stiffness-dependent cellular adaptation. GG16-WT cells were treated with GSK414 for 6 days while growing on hydrogels with increasing stiffness. ( A ) Cells were stained for FLNA and F-Actin and imaged with confocal microscopy. ( B - D ) Quantified FLNA, F-Actin expression, and cell elongation. * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 ATF4 is not involved in cellular adaption to matrix stiffness. GG16-WT and GG16-ATF4-KO cells were cultured on hydrogels with increasing stiffness. ( A , E ) Cells were stained for FLNA, F-Actin, and nuclei and imaged with confocal microscopy. Quantified expression of FLNA and F-Actin ( B , C , F , G ) and cell elongation ( D , H ). * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 4 miR-200c regulates migration associated genes such as filamin A. (a) A proteomic analysis of a TALENs knock-out (KO) of miR-200c in MCF7 cells revealed a set of proteins with differential expression, of which 50% are involved in migratory processes and are shown in the table. (RT qPCR showed that after adding 5 mug / ml doxycycline for 48 h the expression of FLNA mRNA (b) as well as filamin A protein(c) (normalized to tubulin) decreased significantly in MDA-MB 231 TRIPZ 200c cells. (d) Immunofluorescence staining of filamin A (green) and DAPI (blue) in MCF7 Ctrl and KO 200c showed significantly increased relative intensity of filamin A, in contrast to (e) the MDA-MB-231 cells which showed a strong decrease in filamin A intensity after induction of miR-200c (all N = 3; error bars indicate standard deviation SD; * p

MA5-11705

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