Anti-SH3PX1/SNX9 antibody [EPR14399]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

Immunofluorescent analysis of acetone-fixed HeLa cells labeling SH3PX1/SNX9 with ab181856 at 1/100 dilution followed by Goat anti rabbit IgG (Alexa Fluor®488) secondary antibody at 1/200 dilution. Counter stained with Dapi.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

Intracellular flow cytometric analysis of 2%paraformaldehyde-fixedHeLa cells labeling SH3PX1/SNX9 with ab181856 at 1/10 dilution (red) compared to a Rabbit IgG monoclonal isotype control (green), followed by Goat anti rabbit IgG (FITC) secondary antibodyat 1/150 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

Immunofluorescent analysis of 4% paraformaldehyde-fixed HCT-116 cells labeling SH3PX1/SNX9 with ab181856 at 1/100 dilution followed by Goat anti rabbit IgG (Alexa Fluor®488) secondary antibody at 1/200 dilution. Counter stained with Dapi.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling SH3PX1/SNX9 with ab181856 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

Western blot analysis of HepG2 cell lysate immunoprecipitated using ab181856 at 1/50 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate secondary antibody was used at 1/1000 dilution.

All lanes:

Immunoprecipitation - Anti-SH3PX1/SNX9 antibody [EPR14399] (ab181856)

Predicted band size: 67 kDa

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• WB

Lab

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : SH3PX1/SNX9 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : HCT116 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab181856 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab181856 was shown to specifically react with SH3PX1/SNX9 when SH3PX1/SNX9 knockout samples were used. Wild-type and SH3PX1/SNX9 knockout samples were subjected to SDS-PAGE. ab181856 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (ab181856)

Predicted band size: 67 kDa

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• WB

Supplier Data

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

All lanes:

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (ab181856) at 1/50000 dilution

All lanes:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 67 kDa

Observed band size: 78 kDa

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• WB

Supplier Data

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

All lanes:

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (ab181856) at 1/10000 dilution

All lanes:

HCT-116 cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 67 kDa

Observed band size: 78 kDa

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• WB

CiteAb

Western blot - Anti-SH3PX1/SNX9 antibody [EPR14399] (AB181856)

SH3PX1/SNX9 western blot using anti-SH3PX1/SNX9 antibody [EPR14399] ab181856. Publication image and figure legend from Jarsch, I. K., Gadsby, J. R., et al., 2020, J Cell Biol, PubMed 32328641.

ab181856 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 ab181856 please see the product overview.

SNX9 in cells.(A) Single frame from a TIRFM time-lapse of Xenopus chordamesoderm cells expressing mNeonGreen-SNX9 (green) and GAP-RFP membrane marker (magenta) showing SNX9 at the tip of filopodia. Scale bar, 10 μm. (B) Western blots against SNX9 or the loading control DM1α (tubulin) of untreated RPE-1 cells and cells treated with either non-targeting control (Non-targ.) or anti SNX9 siRNA showing knockdown with the SNX9 siRNA sequence. (C) Quantification of the number of filopodia present per cell in untreated RPE-1 cells and cells treated with either nontargeting control or anti SNX9 siRNA, illustrating no change in the average number of filopodia per cell on SNX9 knockdown. Each data point represents an individual cell; n = 18, 13, and 23 cells for the untreated (unt.), non-targeting control (NT Cont.), and SNX9 siRNA–treated conditions, respectively. Lines indicate mean ± SD. Statistical significance was assessed by Kruskal-Wallis ANOVA with Dunn's multiple comparisons test. Overall ANOVA P = 0.1206 (n.s.), multiple comparisons : untreated vs. nontargeting control P = 0.1481 (n.s.), untreated vs. SNX9 siRNA P > 0.9999 (n.s.), nontargeting control vs. SNX9 siRNA P = 0.2800 (n.s.). (D and E) Example images of fluorescence confocal images of HeLa cells either (D) uninfected or (E) infected with C. trachomatis (yellow) and immunolabeled using mouse anti-human SNX9 (purple) and phalloidin (green), illustrating SNX9 presence in filopodia and how C. trachomatis stick to preexisting filopodia that contain SNX9 in either the tip or shaft of the filopodia. Scale bars, 2 μm. (F) Quantification of SNX9 presence in filopodia in both uninfected HeLa cells and in cells infected with C. trachomatis. SNX9 immunolabeling stains ~95% of filopodia in untreated RPE-1 with no difference on C. trachomatis infection. Each data point represents an individual imaging region; n = 20 regions per condition. Lines indicate mean ± SD. Infected : 2,080 filopodia from 97 cells; uninfected : 1,648 filopodia from 126 cells. Statistical significance was assessed by unpaired t test, P = 0.3239 (n.s.).

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