- PA5-53295 - Invitrogen Antibodies STK4 antibody

Guo F, Wang W, Song Y, Wu L, Wang J, Zhao Y, Ma X, Ji H, Liu Y, Li Z, Qin G
Cell cycle (Georgetown, Tex.) 2020 Aug;19(16):1997-2006

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MST1 in Caco-2 cells transfected with control siRNA, target specific siRNA probe #1, using a MST1 Polyclonal Antibody (Product # PA5-53295). Remaining relative intensity is presented. Loading control: Anti-GAPDH.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MST1 in human cell line CACO-2 using a MST1 Polyclonal Antibody (Product # PA5-53295).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MST1 in mouse cell line NIH-3T3 and rat cell line NBT-II using a MST1 Polyclonal Antibody (Product # PA5-53295).

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 # PA5-53295, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of STK4 was achieved by transfecting Hep G2 with STK4 specific siRNAs (Silencer® select Product # s13571, s13572). Western blot analysis (Fig. a) was performed using Whole cell extracts from the STK4 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with MST1 (STK4) Polyclonal Antibody (Product # PA5-53295, 0.1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to STK4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-MST1 (STK4) Polyclonal Antibody (Product # PA5-53295) and a 55 kDa band corresponding to STK4 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2), Jurkat (Lane 3), A-431 (Lane 4) and Hep G2 (Lane 5) were electrophoresed using NuPAGE™ 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 the primary antibody (0.1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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)
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Experimental details: Immunofluorescent staining of MST1 in human cell line CACO-2 shows positivity in nucleus but excluded from the nucleoli. Samples were probed using a MST1 Polyclonal Antibody (Product # PA5-53295).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of MST1 in human cell line CACO-2 using a MST1 Polyclonal Antibody (Product # PA5-53295) shows localization to nucleoplasm, nuclear bodies and cytosol.

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 2% BSA for 45 minutes at room temperature. The cells were labeled with MST1 Polyclonal Antibody (Product # PA5-53295) at 2 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), 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 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: Immunohistochemical staining of MST1 in human lymph node shows strong cytoplasmic positivity in cells outside reaction centra. Samples were probed using a MST1 Polyclonal Antibody (Product # PA5-53295).

PA5-53295