mouse anti-c-abl  (Millipore)

Journal: Cell Death & Disease

Article Title: Anti-apoptotic HAX-1 suppresses cell apoptosis by promoting c-Abl kinase-involved ROS clearance

doi: 10.1038/s41419-022-04748-2

Figure Lengend Snippet: A Lysates from MCF-7 cells were immunoprecipitated with anti-c-Abl antibody and rabbit IgG. The precipitates were fractionated with SDS-PAGE and immunoblotted with anti-HAX-1 and anti-c-Abl antibodies. Whole lysates (2% v/v) were used as controls to confirm HAX-1 and c-Abl expression. B HEK 293 cells were co-transfected with Myc-HAX-1 and Flag-c-Abl or Flag-vector as a control. Cell lysates were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were evaluated by SDS-PAGE and immunoblotting and probed with anti-Myc or anti-Flag antibodies. C HEK 293 cells were co-transfected with indicated plasmids. To normalize the input level of c-Abl, four-fold amount of Myc-c-Abl expressing plasmid was used in Flag-HAX-1 co-transfection than that in Flag-Vector co-transfection. Cell lysates were analyzed by immunoprecipitation and immunoblotting. D Lysates from HEK 293 cells transfected with Flag-HAX-1 were incubated with equal amounts of Sepharose beads conjugated to GST or the GST-c-Abl-SH3/SH2 fusion protein. The absorbates were analyzed by Western blot. Whole lysate (2% v/v) was included as a control. Staining with Coomassie brilliant blue confirmed the presence of GST and the GST-c-Abl-SH3/SH2 fusion protein. E Lysates from HEK 293 cells transfected with indicated plasmids were subjected to immunoprecipitation with anti-Flag and SDS-PAGE and subsequently analyzed by immunoblotting. F Lysates from HEK 293 cells transfected with indicated plasmids were incubated with GST or GST-c-Abl-SH3 fusion protein. The adsorbates were analyzed by Western blot with anti-Myc antibody. G HEK 293 cells were transfected as indicated. To normalize the input level c-Abl, four fold amount of Flag-c-Abl expressing plasmid was used in Myc-HAX-1 co-transfection than that in Myc-HAX-1(Q190X) co-transfection. Lysates were subjected to immunoprecipitation with anti-Flag antibody and subsequently analyzed by Western blot.

Article Snippet: The fixed cells were incubated with rabbit anti-HAX-1 and mouse anti-c-Abl (Sigma-Aldrich) primary antibodies.

Techniques: Immunoprecipitation, SDS Page, Expressing, Transfection, Plasmid Preparation, Western Blot, Cotransfection, Incubation, Staining

Journal: Cell Death & Disease

Article Title: Anti-apoptotic HAX-1 suppresses cell apoptosis by promoting c-Abl kinase-involved ROS clearance

doi: 10.1038/s41419-022-04748-2

Figure Lengend Snippet: A An in situ proximity ligation assay (in situ PLA) was used for the detection of HAX-1 and c-Abl binding complexes. MCF-7 cells treated with or without CDDP (25 mM, 8 h) or H 2 O 2 (1 mM, 3 h) were incubated with target primary antibodies from two different species or with anti-c-Abl antibody as a control. The red spots reveal c-Abl/HAX-1 interaction. Nuclei are stained with Hoechst 33342. Slides were evaluated using an LSM 510 META confocal microscope (Carl Zeiss). Cell images obtained were exported using the Zeiss LSM Image Browser (Carl Zeiss) in TIF format for further analysis. B Quantification of HAX-1-c-Abl interaction complexes. The number of complexes per cell was counted in at least three fields. Quantifications were given as the mean±S.D. Representative results are shown from experiments repeated three times. *** p < 0.001, Student’s t test. C MCF-7 or HAX-1 siRNA cells treated with the indicated dosage of H 2 O 2 were subjected to in situ PLA analysis. D Quantification of HAX-1-c-Abl interaction complexes. The number of complexes per cell was counted in at least three fields. Quantifications were given as the mean ± S.D. Representative results are shown from experiments repeated three times. n.s., not significant; ** p < 0.01, *** p < 0.001, Student’s t test. E MCF-7 cells treated with or without CDDP (25 mM, 8 h) were incubated with target primary anti-c-Abl and anti-HAX-1 antibodies and then incubated with FITC- or TRITC-linked secondary antibodies. Nuclei are stained with Hoechst 33342 (left). The relative colocalization ratio of HAX-1 with c-Abl was analyzed by ImageJ software (right). At least 15 cells were analyzed, and the data were shown as mean±S.D. * p < 0.05, Student’s t test. F Lysates prepared from MCF-7 cells treated with or without H 2 O 2 (1 mM, 3 h) were analyzed by immunoprecipitation and immunoblotting.

Article Snippet: The fixed cells were incubated with rabbit anti-HAX-1 and mouse anti-c-Abl (Sigma-Aldrich) primary antibodies.

Techniques: In Situ, Proximity Ligation Assay, Binding Assay, Incubation, Staining, Microscopy, Software, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Anti-apoptotic HAX-1 suppresses cell apoptosis by promoting c-Abl kinase-involved ROS clearance

doi: 10.1038/s41419-022-04748-2

Figure Lengend Snippet: A HEK 293 cells were co-transfected with Flag-c-Abl and Myc-HAX-1 or Myc-vector. Cell lysates were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were normalized by c-Abl level and immunoblotted with indicated antibodies. B In vitro immune complex kinase assay. Flag-c-Abl and Flag vectors were transfected with or without Myc-HAX-1 into HEK 293 cells as indicated. Proteins were purified using anti-Flag antibody-coupled Sepharose beads and then eluted with Flag peptide. In the kinase assay reaction system, the purified recombinant fusion proteins were incubated with GST-Crk fusion protein or GST protein at 30 °C for 30 min. ATP was added to the reaction buffer as indicated. The products were analyzed by SDS-PAGE and Western blot with indicated antibodies. C MCF-7 scramble and HAX-1 knockdown cell lines (MCF-7/siHAX-1) were transfected with Flag-Abl vector. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by Western blot with indicated antibodies. The immunoprecipitates were normalized by c-Abl level. D The MCF-7 scramble or HAX-1 knockdown cell line (MCF-7/siHAX-1) was transfected with Myc-HAX-1 or vector control. Before being harvested, cells were treated with the indicated dosage of H 2 O 2 for 3 h. The cell lysates were analyzed by immunoprecipitation and immunoblotted. The immunoprecipitates were normalized by c-Abl level. E The MCF-7 scramble or HAX-1 knockdown cell line (MCF-7/siHAX-1) were subjected to 10 Gy irradiation. Cell lysates were immunoprecipitated with anti-c-Abl antibody, and immunoblots were probed with indicated antibodies. The immunoprecipitates were normalized by c-Abl level. F Flag-c-Abl was co-transfected with Myc-HAX-1 or Myc-HAX-1(Q190X) in 293 cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were normalized by c-Abl level and immunoblotted with indicated antibodies. G HEK 293 cells were transfected with Myc-HAX-1, Myc-HAX-1(Q190X) or Myc-vector. Cell lysates were immunoprecipitated with anti-c-Abl antibody. The immunoprecipitates were normalized by c-Abl level and immunoblotted with indicated antibodies. H MCF-7 cells transfected with Myc-HAX-1 or Myc-vector plasmids were incubated with target primary antibodies from two different species or with anti-c-Abl antibody as a control. The red spots reveal c-Abl/HAX-1 interaction. Nuclei are stained with Hoechst 33342 (left). Quantification of HAX-1-c-Abl interaction complexes. The number of complexes per cell was counted in at least three fields. Quantifications were given as the mean±S.D. Representative results are shown from experiments repeated three times (right). *** p < 0.001, Student’s t test.

Article Snippet: The fixed cells were incubated with rabbit anti-HAX-1 and mouse anti-c-Abl (Sigma-Aldrich) primary antibodies.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Immune Complex Kinase Assay, Purification, Kinase Assay, Recombinant, Incubation, SDS Page, Western Blot, Irradiation, Staining

Journal: Cell Death & Disease

Article Title: Anti-apoptotic HAX-1 suppresses cell apoptosis by promoting c-Abl kinase-involved ROS clearance

doi: 10.1038/s41419-022-04748-2

Figure Lengend Snippet: A Western blot analysis with specific antibodies for determination of the levels of total Flag-c-Abl status in HEK 293 cells expressing different dosages of Myc-HAX-1 protein. All cells were transfected with 1 μg Flag-c-Abl, and lanes 2, 3 and 4 were transfected with 0.5, 1.0 and 2.0 μg Myc-HAX-1, respectively. Mock (Lane 1) served as a control in which empty vector was used to transfect the cells. Cellular tubulin was used as an internal control for comparison of protein load in each lane. B Western blot analysis of HEK 293 cells transfected with the indicated plasmids. The expression of β-actin served as a loading control. C Western blot using anti-HAX-1 antibody identified the MCF-7 clones that were transfected with the HAX-1 -specific siRNA or scrambled sequence. The expression level of HAX-1 and c-Abl was normalized to the tubulin loading control. D HEK 293 cells were treated with increased concentrations of H 2 O 2 for 3 h and subjected to Western blot analysis. E Flag-c-Abl was co-expressed with the Myc-vector or with Myc-HAX-1 in HEK 293 cells. Cells were pulsed with [ 35 S] methionine for 45 min, washed and then incubated in [ 35 S] methionine-free DMEM for the indicated time. Lysates were immunoprecipitated with anti-Flag antibody and analyzed by SDS-PAGE and autoradiography. The half-life of Flag-c-Abl was calculated according to the intensity of the signals of three independent experiments. F Flag-c-Abl was transfected into HEK 293 cells with or without Myc-HAX-1 co-expression. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-c-Cbl or anti-Flag antibody. G MCF-7 scramble cells were transfected with Myc-HAX-1, and HAX-1 knockdown cells were transfected with a Myc-HAX-1 rescue construct (Muta). For the ubiquitination assay, cells were treated with MG132 (10 μM) 12 h before harvesting. Cell lysates were immunoprecipitated with anti-c-Abl antibody or IgG as a control. The precipitates were analyzed by Western blot using indicated antibodies. H MCF-7 cells transfected with or without HAX-1 were IR treated (10 Gy) as indicated. Cell lysates were immunoprecipitated with anti-c-Abl antibody and immunoblotted using indicated antibodies. I MCF-7 scramble and two HAX-1 knockdown clones overexpressing Myc-Ub were IR (10 Gy) treated. After 2 h, cells were harvested, and cell lysates were immunoprecipitated with anti-Myc antibody. The ubiquitination levels of the precipitates were assessed by Western blot.

Article Snippet: The fixed cells were incubated with rabbit anti-HAX-1 and mouse anti-c-Abl (Sigma-Aldrich) primary antibodies.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Clone Assay, Sequencing, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Construct, Ubiquitin Assay