- PA5-28765 - Invitrogen Antibodies HUNK antibody

Submitted references HUNK Phosphorylates Rubicon to Support Autophagy.

Zambrano JN, Eblen ST, Abt M, Rhett JM, Muise-Helmericks R, Yeh ES
International journal of molecular sciences 2019 Nov 19;20(22)

Integral membrane protein 2A inhibits cell growth in human breast cancer via enhancing autophagy induction.

Zhou C, Wang M, Yang J, Xiong H, Wang Y, Tang J
Cell communication and signaling : CCS 2019 Aug 22;17(1):105

Staurosporine, an inhibitor of hormonally up-regulated neu-associated kinase.

Zambrano JN, Williams CJ, Williams CB, Hedgepeth L, Burger P, Dilday T, Eblen ST, Armeson K, Hill EG, Yeh ES
Oncotarget 2018 Nov 13;9(89):35962-35973

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using HUNK Polyclonal Antibody (Product # PA5-28765). Sample (30 µg of whole cell lysate). Lane A: A431 . 7.5% SDS PAGE. HUNK Polyclonal Antibody (Product # PA5-28765) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of HUNK in paraformaldehyde-fixed A549 cells using a HUNK polyclonal antibody (Product # PA5-28765) at a 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded OVCA xenograft, using HUNK (Product # PA5-28765) antibody at 1:100 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 ITM2A interacts and is phosphorylated by HUNK. ( a ) Coomassie brilliant blue staining showed that the peptide ""VAIKVIDKKRAKKDTYVTKNLRREGQIQQMI"" existed in the 70 kDa-100 kDa band. ( b ) Co-immunoprecipitation showed the interaction between ITM2A and HUNK in HEK293T cells. ( c ) Flag-HUNK were obtained from the immunoprecipitate in HEK293T cell lysates using Flag antibody. The Flag-HUNK was incubated with the in vitro purified GST-ITM2A WT or T35A protein at 30 degC for 30 min with or without ATP addition, and then the reaction products were subjected to western blotting. ( d ) Flag-ITM2A was obtained from the immunoprecipitate in SKBR-3 cell lysates with or without HUNK knockdown using Flag antibody. Total phosphorylated threonine was detected in the western blotting assay. ( e ) SKBR-3 cells transfected with HA-ITM2A or Flag-HUNK were starved for the indicated times, and HA-ITM2A was immunoprecipitated using HA antibody and used for western blotting analysis. Purified Flag-HUNK was used in the in vitro kinase assay using purified MBP protein as a substrate. ( f ) SKBR-3 cells were transfected with wild-type ITM2A and its T35A mutant simultaneously with or without HUNK siRNA co-transfection for 48 h. Cell lysates were obtained and used for detection by western blotting with the indicated antibodies. ( g ) HEK293T cells were transfected with empty vector and Flag tagged HUNK simultaneously with or without ITM2A siRNA co-transfection for 48 h. Cell lysates were obtain

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Hormonally Upregulated Neu-associated Kinase (HUNK) activity is required for HUNK-mediated autophagy. ( A ) 293T cells were transfected with empty vector, HA-HUNK, or HA-K91M HUNK. Cells were then serum deprived and treated with 100 uM CQ for 4 h, lysed and analyzed for LC3B by immunoblot analysis. ( B ) 293T cells plated on coverslips were transfected with RFP-LC3B and either GFP-HUNK or GFP-K91M-HUNK. Cells were serum deprived and treated with 100 uM CQ for 4 h, followed by fixation and imaging. Quantitation of RFP-LC3B puncta in cells. N >= 3 fields per experiment. Data represent 3 or more experiments. Student's T -test was used to perform statistical analysis. ( C ) Representative images of quantitation in ( B ). Scale bar size = 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 HUNK binds the Beclin-1 complex members. HA-HUNK and either ( A ) Flag-Beclin-1, ( B ) GFP-Atg14L, ( C ) Flag-Vps34, ( D ) Flag-UVRAG, or ( E ) Flag-Rubicon were co-transfected into 293T cells. HUNK was immunoprecipitated and binding of individual proteins was assessed via immunoblotting for each Beclin-1 complex member. Each member of the Beclin complex co-immunoprecipitated with HUNK. HUNK was immunoprecipitated and detected with anti-HA antibody and Beclin-1 complex members were detected with anti-Flag or anti-GFP antibodies. Interactions were also detected by individual antibodies to each Beclin-1 complex member.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 HUNK phosphorylates Rubicon ( A ) In vitro HUNK kinase assay using recombinant Rubicon (aa 1-375) as substrate. HUNK was preincubated with either DMSO or the HUNK inhibitor staurosporine (STU, 5 uM). ( B ) HA-HUNK or HA-K91M HUNK and Flag-Rubicon were expressed in 293T cells. Flag-Rubicon was immunoprecipitated using anti-Flag affinity resin and isolated protein was immunoblotted using anti-pSer and anti-pSer/Thr antibodies.

PA5-28765

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