Anti-DCAMKL1 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-DCAMKL1 antibody (AB31704)

ab31704 staining DCAMKL1 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab31704 at 5 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DCAMKL1 antibody (AB31704)

ICC/IF image of ab31704 stained PC12 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab31704, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue).

• IP

Unknown

Immunoprecipitation - Anti-DCAMKL1 antibody (AB31704)

DCAMKL1 was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to DCAMKL1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31704.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 82kDa : DCAMKL1; non specific - 52 and 27kDa : We are unsure as to the identity of this extra band.

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Immunoprecipitation - Anti-DCAMKL1 antibody (ab31704)

Predicted band size: 82 kDa

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• WB

Project

Western blot - Anti-DCAMKL1 antibody (AB31704)

The 82 kDa and 47 kDa bands correspond to the AL and BL isoforms respectively. DCAMLK1 has a number of potential phosphorylation sites which may explain the higher migrating bands at 52 and 54 kDa.

All lanes:

Western blot - Anti-DCAMKL1 antibody (ab31704) at 1 µg/mL

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Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 82 kDa

Observed band size: 30 kDa,47 kDa,52 kDa,54 kDa,82 kDa

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Exposure time: 5min

• WB

Lab

Western blot - Anti-DCAMKL1 antibody (AB31704)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : DCAMKL1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : NIH3T3 whole cell lysate (20 μg)
Lane 4 : Human brain whole tissue lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab31704 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab31704 was shown to specifically react with DCAMKL1 in wild-type HAP1 cells along with additional cross reactive bands. No bands were observed when DCAMKL1 knockout cells were examined. Wild-type and DCAMKL1 knockout samples were subjected to SDS-PAGE. ab31704 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-DCAMKL1 antibody (ab31704)

Predicted band size: 82 kDa

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• WB

Project

Western blot - Anti-DCAMKL1 antibody (AB31704)

The 82 kDa and 47 kDa bands correspond to the AL and BL isoforms respectively. This antibody should not detect the AS and BS isoforms of DCAMLK1.

All lanes:

Western blot - Anti-DCAMKL1 antibody (ab31704) at 1 µg/mL

All lanes:

Mouse Brain Whole Tissue Lysate at 20 µg

Secondary

All lanes:

IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Predicted band size: 82 kDa

Observed band size: 47 kDa,82 kDa

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• WB

Project

Western blot - Anti-DCAMKL1 antibody (AB31704)

All lanes:

Western blot - Anti-DCAMKL1 antibody (ab31704) at 1 µg/mL

All lanes:

Brain (Rat) Tissue Lysate - normal tissue at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 82 kDa

Observed band size: 47 kDa,82 kDa

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• WB

AbReview81447****

Western blot - Anti-DCAMKL1 antibody (AB31704)

All lanes:

Western blot - Anti-DCAMKL1 antibody (ab31704) at 1 µg/mL

Lane 1:

HCT116 WT at 30 µg

Lane 2:

MiaPaCa2, at 30 µg

Lane 3:

SW480 at 30 µg

Lane 4:

SW620 at 30 µg

Lane 5:

HT-29 at 30 µg

Lane 6:

H4 at 30 µg

Lane 7:

mouse brain lysate at 30 µg

Secondary

All lanes:

IRDye® 800CW Goat Anti-Rabbit, at 1/20000 dilution

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This image is courtesy of an anonymous Abreview

• Flow Cyt

CiteAb

Flow Cytometry - Anti-DCAMKL1 antibody (AB31704)

Flow cytometry/Cell sorting using Anti-DCAMKL1 antibody, ab31704. Publication image from Zhou, L. et al., 2021, Nat Commun, 34294718. Legend direct from paper.

IL-22 suppresses IL-13-induced tuft cell differentiation.a Analysis of Dclk1 and Trpm5 expression by qRT-PCR in enteroid culture (n = 3) (mean ± SD) (Dclk1, ***P = 0.0008; Trpm5, ****P < 0.0001). Representative data of three independent experiments. b DCLK1 expression in enteroid culture with indicated treatment (IL-13 (50 ng/ml), IL-17A (100 ng/ml), and/or IL-22 (5 ng/ml)) measured by flow cytometry. Representative data of two independent experiments. c Percentages of DCLK1+ cells in EpCAM+ cells (mean ± SD) (n = 3) (none vs. IL-13, ****P < 0.0001; IL-13 vs. IL-13 + IL-22, **P = 0.0045). Compiled data from one experiment. d Confocal analysis of DCLK1 expression in enteroid culture with indicated treatment. Representative data of two independent experiments. Green, DCLK1. Blue, DAPI. Scale bar : 50 µm. e Left panel, picture of the small intestine of 8-week-old littermate Il22Cre/+ and Il22Cre/Cre mice (*P = 0.0223). Representative data of two independent experiments. Right panel, small intestine length in mice with indicated genotypes (n = 3). Compiled data from two independent experiments. f IL-4, IL-5, and IL-13 expression in ILC2s or CD4+ T cells by flow cytometry from the small intestine of 8-week-old Il22Cre/+ and Il22Cre/Cre mice (n = 3) (ILC2 : IL-4, **P = 0.0076; IL-5, **P = 0.0024; IL-13, *P = 0.0245; CD4+ T : IL-4, P = 0.8613; IL-5, P = 0.4343; IL-13, P = 0.2181). Compiled data from two independent experiments. g Analysis of Dclk1, Trpm5, Gnat3, and Il25 expression by qRT-PCR in small intestine tissues of 8-week-old Il22Cre/+ and Il22Cre/Cre mice (n = 3) (Dclk1, ****P < 0.0001; Trpm5, **P = 0.0023; Gnat3, ****P < 0.0001; Il25, **P = 0.0062). Representative data of two independent experiments. h, iTfamfl/flRorc-cre mice at the age of 6 weeks old received hydrodynamic tail vein injection of either empty or IL-22 vectors. Mice were sacrificed at 10 weeks of age and small intestine tissues were harvested. h qRT-PCR gene expression analysis of small intestine tissue IL-22-target gene Reg3g, type 2 cytokine genes Il4, Il5, and Il13, and tuft cell-associated genes Dclk1, Trpm5, Gnat3, and Il25 (n = 3) (Reg3g, ***P = 0.0001; Il4, *P = 0.0139; Il5, ***P = 0.0004; Il13, ***P = 0.0001; Dclk1, ****P < 0.0001; Trpm5, ****P < 0.0001; Gnat3, **P = 0.0011; Il25, **P = 0.0063). Representative data of three independent experiments. i Top panel, picture of the small intestine length. Bottom panel, quantification of the small intestine length (n = 4 for empty group and n = 3 for IL-22 treatment group) (***P = 0.0009). Compiled data from one experiment. Data are shown as mean ± SD in a, c, e–i.

• WB

CiteAb

Western blot - Anti-DCAMKL1 antibody (AB31704)

Western Blotting using Anti-DCAMKL1 antibody, ab31704. Publication image from Ali, N. et al., 2014, Mol Cancer, 24885928. Legend direct from paper.

LRRK2-IN-1 inhibits DCLK1 kinase activity. An in vitro kinase assay was performed using Purified active DCLK1 kinase (0.25 µg) with 2.5 µg of autocamtide II substrate, 1 µM ATP, and either DMSO, 0.6, 2.5, 5, 10, or 50 nM LRRK2-IN-1 (A). Using relative luminescent units (RLU) data, a sigmoidal-dose response curve was plotted in GraphPad Prism 6.0 (adj. R2 = 0.952) revealing an IC50 value of 2.61 nM (B). AsPC-1 cells were treated with LRRK2-IN-1 at varying concentrations for 48 h. Following treatment cells were lysed, protein was isolated and quantified by BCA assay, and immunoblotting was performed withα-phospho-DCLK1. The ratio of phospho-DCLK1 to total DCLK1 (Figure 4B; 48 h) was determined and demonstrated decreased phosphorylation of DCLK1 (p < 0.05) following treatment (C). Schematic demonstrating the shared protein kinase domain between DCLK1 isoforms referenced in Uniprot [Swiss-Prot : O15075] (D). Three dimensional view of LRRK2-IN-1 binding site in DCLK-long-β revealing predicted interactions with residues of the hinge region, catalytic loop (C-loop), activation loop (A-loop),αC-helix (αC), and the highly conserved lysine (Lys112) of the kinase catalytic domain suggesting that LRRK2-IN-1 competes with ATP for the DCLK1 kinase binding pocket. Dashed lines mark the hydrogen bond formed with the conserved aspartate (“D” of the “DFG” motif) of the activation loop (E).

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