- PA5-22015 - Invitrogen Antibodies STK4 antibody

Zhang L, Han P
Journal of musculoskeletal & neuronal interactions 2022 Sep 1;22(3):411-421

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MST1 using 50 µg of mouse brain lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a MST1 polyclonal antibody (Product # PA5-22015) at a dilution of 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MST1 using 30 µg of HepG2 lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a MST1 polyclonal antibody (Product # PA5-22015) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of STK4 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR879788_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of STK4 was performed by loading 30 µg of Caco-2 Wild Type (Lane 1), Caco-2 Cas9 (Lane 2) andCaco-2 STK4 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with a MST1 (STK4) Polyclonal Antibody (Product # PA5-22015, 1:1,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to STK4. An uncharacterised band was observed at ~160 kDa in all the samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of MST1 was performed by separating 50 µg of Mouse tissue extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a MST1 Polyclonal Antibody (Product # PA5-22015) at a dilution of 1:2000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), Jurkat (Lane 2), HEK-293 (Lane 3), HEK-293 treated with Staurosporine (0.5uM for 3h) (Lane 4) and A-431 (Lane 5). The blot was probed with Anti-MST1 Polyclonal Antibody (Product # PA5-22015, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 57kDa band corresponding to MST1 was observed across all the cell lines tested. A cleaved fragment was observed to be induced upon treatment of HEK-293 cells with Staurosporine. An uncharacterized band was observed at 160kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MST1 in paraformaldehyde-fixed U2OS cells using a MST1 polyclonal antibody (Product # PA5-22015) (Green) at a 1:500 dilution. Alpha-tubulin filaments were labeled with Product # PA5-29281 (Red) at a 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Confocal immunofluorescence analysis (Olympus FV10i) of paraformaldehyde-fixed U2OS, using STK4 antibody (Product # PA5-22015) (Green) at 1:500 dilution. Alpha-tubulin filaments were labeled with (Product # MA1-25054) (Red) at 1:2,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of MST1 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with MST1 Polyclonal Antibody (Product # PA5-22015) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 nucleus and cytoplasmic localization. Panel e 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: Immunohistochemistry (Paraffin) analysis of MST1 was performed in paraffin-embedded human lung adenocarcinoma tissue using MST1 Polyclonal Antibody (Product # PA5-22015) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded Cal27 xenograft, using MST1 (Product # PA5-22015) antibody at 1:500 dilution. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: STK4 antibody immunoprecipitates STK4 protein in IP experiments. IP samples: HepG2 whole cell extract. A. 50 µg HepG2 whole cell extract. B. Control with 4 µg of preimmune Rabbit IgG. C. Immunoprecipitation of STK4 protein by 4 µg STK4 antibody (Product # PA5-22015). 7.5 % SDS-PAGE. The immunoprecipitated STK4 protein was detected by STK4 antibody (Product # PA5-22015) diluted at 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: MiR-374-5p activates autophagy of injured neurons through directly targeting STK-4. (A) Bioinformatics prediction of binding sites between miR-374-5p and the 3'UTR of the STK-4 mRNA. (B) The interaction of miR-374-5p and the 3'UTR of the STK-4 mRNA was examined by dual-luciferase reporter assay. (C) The expression level of STK-4 was detected by western blot. Cell apoptosis was examined by TUNEL staining (D), flow cytometry assay (E), and western blot of apoptosis markers (F). The autophagy flux was examined by immunofluorescence staining (G) and western blot of autophagy flux markers (H). The band intensities were analyzed using the gray values in Image J software.

PA5-22015