Anti-p50 dynamitin antibody [EPR5095]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-p50 dynamitin antibody [EPR5095] (AB133492)

Immunofluorescence analysis of p50 dynamitin in SH-SY5Y cells using ab133492 at a dilution of 1/100.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p50 dynamitin antibody [EPR5095] (AB133492)

Immunohistochemical analysis of paraffin-embedded Human cerebrum  tissue labelling p50 dynamitin with ab133492 at 1/400 dilution followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) at Ready to use dilution. Positive staining on human cerebrum. The section was incubated with ab133492 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection) at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

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Western blot - Anti-p50 dynamitin antibody [EPR5095] (AB133492)

All lanes:

Western blot - Anti-p50 dynamitin antibody [EPR5095] (ab133492) at 1/1000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

A375 cell lysate at 10 µg

Lane 3:

Saos2 cell lysate at 10 µg

Lane 4:

SH SY5Y cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 44 kDa

Observed band size: 44 kDa

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• WB

CiteAb

Western blot - Anti-p50 dynamitin antibody [EPR5095] (AB133492)

p50 dynamitin western blot using anti-p50 dynamitin antibody [EPR5095] ab133492. Publication image and figure legend from Dwivedi, D., Kumari, A., et al., 2019, J Cell Biol, PubMed 30674580.

ab133492 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 ab133492 please see the product overview.

Hook2 acts as a dynein–dynactin linker. (A) Domain architecture of Hook2 and its domain deletion fragments/mutants used in the study. (B) GST or GST-tagged LIC1 (389–523 aa) bound to glutathione beads were incubated with MBP-tagged Hook2 N427 (WT, Q143A, and I150A), and immunoblotted (IB) with an anti-MBP antibody for Hook2 (WT/mutants). LIC1 in the pelleted beads was detected using Ponceau S staining of the membrane. The asterisk indicates BSA protein band used for blocking glutathione beads. (C) Ratio of band intensity of pulldown to input Hook2 fragment signals in B (n = 3). (D) HEK293T cell lysates were incubated with MBP alone or MBP-tagged Hook2 N427 (WT, Q143A, and I150A) bound to amylose beads, and IB for DIC and p150glued. The amount of recombinant Hook2 (WT/mutants) protein was analyzed by Coomassie staining. (E) Ratio of band intensity of pulldown to input Hook2 (WT/mutants) signal in D (n = 3). (F) Protein-A/G beads bound to control IgG or anti-Hook2 antibody were incubated with HEK293T lysates; the interactome IP was IB to check the presence of different dynein subunits. (G) Protein-A/G beads bound to antibodies against DIC, p150glued, Arp1, and p50/dynamitin were incubated with HEK293T lysate; the interactome IP was IB to check the presence of Hook2. (H) Lysates from HEK293T cells treated with control or Hook2 siRNA and transfected with indicated plasmids were incubated with protein-G beads bound to antibodies against DIC and p150glued, and IP were IB with the indicated antibodies. Arrows mark Hook2 (WT) transfected lanes. (I) Ratio of normalized band intensity (EV) of IP DIC to p150glued and vice versa in H (n = 2). (J) Representative images of FRB-FKBP12-rapamycin dimerization assay in fixed HeLa cells. Bars, 10 μm. (K and L) Mitochondrial distribution quantified as intensity with respect to relative distance from the nucleus (n = 3; 10 cells/experiment). Data represent mean ± SD (***, p < 0.001; Student's t test).

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