PA5-17167

antibody from Invitrogen Antibodies

Targeting: SHTN1 KIAA1598, shootin-1, shootin1

Immunoprecipitation

Antibody data

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Validation data

SHTN1 protein structure - PA5-17167 shown in red.

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 6 CDKL5 influences shootin1 phosphorylation in primary cortical neurons. (A) Total cell extracts of DIV7 cortical neurons were treated with or without lambda phosphatase (lamda-PPase) and analyzed by two-dimensional gel electrophoresis and immunoblotting with antibodies against shootin1, beta-actin and, as control for the lamda-PPase treatment, phopho-ERK1/2. (B) Primary cortical neurons were infected with shLacZ- or shCDKL5#1-expressing viral particles at DIV0 and total cell lysates were prepared at DIV7 and subjected to two-dimensional gel electrophoresis. Shootin1 and NFL were detected by immunoblotting; the single NFL-spot was used as internal control for alignment. Silencing of CDKL5 was confirmed by western blot (right panel). (n = 3).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 1 CDKL5 interacts with shootin1 in vivo. (A) A yeast two-hybrid screening identified shootin1 as a CDKL5 interacting protein. The C-terminal region of hCDKL5, spanning amino acids 299-1030, was used as bait (upper, thick bar). The diagram below shows shootin1 with its coiled coil domains in black. The clones identified in the screen are indicated as black bars and the minimum CDKL5 interacting region as a black bar. (B) Coimmunoprecipitation of P5-7 brain lysates with anti-CDKL5 (upper, n = 3) or anti-shootin1 (lower, n = 3) antibodies (both rabbit). IgGs were used as negative control. The immunoprecipitates and inputs (5% of the brain lysates) were analyzed by immunoblotting for CDKL5 and shootin1 (using a goat anti-shootin1 antibody). Asterisks indicate the immunoglobulin heavy chains and the open circle an unspecific band detected with anti-CDKL5. (C) Coimmunoprecipitation of HeLa cells overexpressing either Flag-CDKL5 or shootin1 or both proteins together. Whole cell lysates were immunoprecipiated with an anti-Flag resin and inputs (5%) and immunocomplexes analyzed by western blotting as indicated. Asterisk shows an anti-shootin1 reactive protein that copurifies with CDKL5. (n = 3).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 2 CDKL5 and shootin1 are coexpressed in brains and neurons. (A) Western blot analysis showing CDKL5 and shootin1 levels in mouse brain at the indicated developmental stages using Tuj1 as loading control. (n = 2) (B) Shootin1 is expressed in the cortex, as early as E13, in the cortical plate (cp) and its levels increase ongoing with development (a,b,c,d); Cdkl5 (b',c',d') follow the same pattern. Low but detectable levels of shootin1 and Cdkl5 mRNAs are present in cells migrating out of the ventricular and sub-ventricular zone (vz-svz) towards their final destination in the cortical plate (b,b'). At E18 shootin1 and Cdkl5 are strongly expressed throughout the whole thickness of the cortex (d,d'). Scale bars: 50 mum: b,b',c,c'; 100 mum: d,d'; 200 mum: a. (C) Western blot showing CDKL5 and shootin1 levels in cultured primary hippocampal neurons at the indicated stages. A longer exposure of the 18 h time point is shown to the right. (n = 2). (D) Immunofluorescence analysis (left) of hippocampal neurons at stages 2-3 with antibodies against CDKL5 (green) and shootin1 (red). The small panels show the magnification of the minor processes/axons indicated with asterisks. Quantitative profiles showing the fluorescence intensities of shootin1 (red) and CDKL5 (green) from the soma to the distal tip of the neurites/axons indicated with asterisks are shown to the right. Scale bar: 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 3 CDKL5 knock-down causes aberrant neuronal polarization. (A) Western blot showing CDKL5 and shootin1 levels in primary hippocampal neurons infected with shRNAs against CDKL5, shootin1 or, as control, LacZ. Neurons were infected at DIV0 and cell lysates prepared after 96 h. Tuj1 was used as loading control. The graph shows the quantified protein levels as means of n>=3 experiments, *p