Anti-CLPP antibody [EPR7133]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLPP antibody [EPR7133] (AB124822)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling CLPP with Purified ab124822 at 1 : 50 dilution (2 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control : PBS instead of the primary antibody.Hematoxylin was used as a counterstain.

• IP

Unknown

Immunoprecipitation - Anti-CLPP antibody [EPR7133] (AB124822)

ab124822 (purified) at 1 : 20 dilution (2μg) immunoprecipitating CLPP in K562 whole cell lysate.
Lane 1 (input) : K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10μg
Lane 2 (+) : ab124822 & K562 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab124822 in K562 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 5000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CLPP antibody [EPR7133] (ab124822)

Predicted band size: 30 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLPP antibody [EPR7133] (AB124822)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse kidney tissue sections labeling CLPP with Purified ab124822 at 1 : 50 dilution (2 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control : PBS instead of the primary antibody.Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLPP antibody [EPR7133] (AB124822)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat stomach tissue sections labeling CLPP with Purified ab124822 at 1 : 50 dilution (2 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control : PBS instead of the primary antibody.Hematoxylin was used as a counterstain.

• WB

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Western blot - Anti-CLPP antibody [EPR7133] (AB124822)

All lanes:

Western blot - Anti-CLPP antibody [EPR7133] (ab124822) at 1/1000 dilution

Lane 1:

Fetal muscle lysate at 10 µg

Lane 2:

Saos-2 (Human osteosarcoma cell line) cell lysate at 10 µg

Lane 3:

A431 (Human epidermoid carcinoma cell line) cell lysate at 10 µg

Lane 4:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate at 10 µg

Lane 5:

K562 cell lysate at 10 µg

Lane 6:

A549 (Human lung carcinoma cell line) cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 30 kDa

Observed band size: 26 kDa

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• WB

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Western blot - Anti-CLPP antibody [EPR7133] (AB124822)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-CLPP antibody [EPR7133] (ab124822) at 0.05 µg/mL

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) whole cell lysate at 15 µg

Lane 3:

Mouse brain lysates at 15 µg

Lane 4:

Rat brain lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 30 kDa

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• WB

CiteAb

Western blot - Anti-CLPP antibody [EPR7133] (AB124822)

Western Blotting using Anti-CLPP antibody [EPR7133], ab124822. Publication image from Fujioka, H. et al., 2016, Nat Commun, 27561680. Legend direct from paper.

HV-3 peptide blocks Htt/VCP binding.(a) Sequence of homology between VCP (human, AAI21795) and Htt (human, NP_002102). Amino acids are represented by the one-letter code; stars (*) indicate identical amino acids; Columns ( : ) indicate high similarity between amino acids. (b) Stick drawings of VCP and Htt main domains. Highlighted in the same colours are the two regions of homology between the two proteins, regions HV-1 and HV-3 in Htt and the corresponding regions HV-2 and HV-4 in VCP. (c) HEK293 cells were transfected with Myc-full-length Htt with 23 Q or 73Q (Myc-23Q FL or Myc-73Q FL) for 48 h following treatment with HV-3 or TAT (3 µM per day, each). The total lysates of cells were subjected to IP followed by WB with the indicated antibodies. (d) The total cell lysates were subjected to IP followed by WB in the indicated groups. (e) Gel filtration chromatogram and SDS-PAGE gel of recombinantly expressed and purified full-length mouse VCP/p97 (upper). Equilibrium binding isotherm for VCP titrated against HV-3 peptide at 15 °C (lower). Each downward spike is from a single injection of HV-3 into the sample cell. The heat exchanged during each injection is calculated from the area under the spike and fit to a binding isotherm. The Kd and n for HV-3 binding are 17.9±7 µM and 2.02±0.23, respectively. The values of δH and δS are −2.145±0.65 Kcal mol−1 and 13.97±3.4 cal mol−1 deg−1, respectively. (f) Biotin-conjugated HV-3 or TAT (10 µM, each) was incubated with total lysates of HD cells or YAC128 mouse brains. Immunoprecipitates were analysed by WB with the indicated antibodies. All Blots shown above are representative of three independent experiments. (g) HD cells were treated with TAT or HV-3 (3 µM per day for 3 days), n=3. (h) YAC128 or wild-type mice from 3–6 months of age and (i) R6/2 or wild-type mice from 5 to 9 weeks of age were received either TAT or HV-3 (3 mg kg−1 per day), n=6 mice/group. VCP mitochondrial levels were determined by WB. Loading control : VDAC. Data are mean±s.e.m. (g–i) ANOVA with Holm-Sidak post hoc test.

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• WB

CiteAb

Western blot - Anti-CLPP antibody [EPR7133] (AB124822)

Western Blotting using Anti-CLPP antibody [EPR7133], ab124822. Publication image from Zhao, Y. et al., 2019, Nat Commun, 30914652. Legend direct from paper.

DA1 peptide blocks Drp1/ATAD3A binding. a Sequence of homology between Drp1 (human, NP_036192) and ATAD3A (human, NP_001164006). Columns ( : ) indicate identical amino acids; single dot (·) indicate high similarity between amino acids. b Stick drawings of ATAD3A and Drp1 main domains. Highlighted in red are the two regions of homology between the two proteins, region DA1 in Drp1 and the corresponding region DA2 in ATAD3A. c Mapping DA1 on crystal structure of Drp1 GTPase-GED fusion construct and DA2 on the N-terminus (residues 1–210) of ATAD3A simulated structure. d Upper : HdhQ7 and HdhQ111 cells were treated with TAT or peptide DA1 (1 µM/day for 4 days). Lower : R6/2 or wildtype mice were treated with TAT or DA1 (1 mg/kg/day) from 6 to 12 weeks. The total lysates of cells or mouse striatum were subject to IP analysis. Three independent experiments. e DA1 or TAT was incubated with recombinant proteins in vitro for 30 mins. GST pull down followed by WB analysis was carried out. 3 independent experiments. f Biotin-conjugated DA1 or TAT (10 µM, each) was incubated with total lysates of HdhQ7 and HdhQ111 cells. Immunoprecipitates were analyzed by WB. 3 independent experiments. g HEK293 cells were transfected with ATAD3A-Flag wildtype (WT) or δN50 mutant plasmids followed by TAT or DA1 treatment (1 µM, each) for 48 h. ATAD3A oligomerization was analyzed by WB. Three independent experiments. h Left : HD cells were treated with TAT or DA1 (1 µM/day for 4 days), 4 independent experiments. YAC128 or wildtype mice from 6 months of age (Middle, 6–7 mice/group), and R6/2 or wildtype mice from 12 weeks of age (Right, 4 mice/group) received either TAT or DA1 (1 mg/kg/day). Drp1 mitochondrial levels were determined by WB. i Drp1 oligomerization was analyzed using total striatal lysates of mice (Upper : YAC128-12 months old; Lower : R6/2 mice-12 weeks old). n = 6 mice/group. j Drp1 oligomerization was analyzed by WB at the indicated groups. Three independent experiments. All data are mean ± SME. One-way ANOVA with Tukey’s post-hoc test

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