American Society of Hematology

Figure 1

$Figure 1. Characterization of INF2 as a MAL-interacting protein and analysis of its subcellular distribution in Jurkat cells. (A) Yeast HF7C strain cells were cotransformed with the pAD-GAL4 255C1 cDNA obtained in the yeast 2-hybrid screen and bait constructs encoding either full-length MAL or MAL2 or the indicated D52-like prey proteins as negative controls. Yeast growth on solid media at 30°C was evaluated as follows: –, no growth after 6-8 days; ++, growth after 3-4 days; +++, growth after 1-2 days. Identical results were obtained for all 3 D52-like proteins. (B) Schematic of the INF2-1 proteins expressed in the experiments in this Figure. (C) The 1% Triton-X-100 insoluble (I) membrane fraction of Jurkat cells was separated from the soluble fraction (S) and, after solublization with 60mM octyl-glucoside, was subjected to pull-down analysis with GST alone or fused to the indicated INF2 fragments. The presence of endogenous MAL was determined by immunoblotting with mAb 6D9 (top). The Coomassie blue staining of the GST proteins used is shown (bottom). (D) Jurkat cells transiently transfected (top) or not (bottom) with Cherry-MAL were stained for endogenous INF2 using anti-INF2 antibodies and secondary antibodies coupled to Alexa-488 (top) or for INF2, α-tubulin, and F-actin using antibodies to INF2 and α-tubulin and secondary antibodies coupled to Alexa-488 or Alexa-647 and with TRITC-phalloidin, respectively (bottom). (E) Jurkat cells transiently expressing GFP fused to INF2-1 or INF2-2 were fixed and stained for α-tubulin and F-actin with anti–α-tubulin antibodies followed by secondary antibodies coupled to Alexa-647 and with TRITC-phalloidin, respectively. (F) Jurkat cells transiently expressing GFP fusions of the indicated fragments of INF2-1 were used to analyze the distribution of α-tubulin and F-actin with anti–α-tubulin antibodies followed by secondary antibodies coupled to Alexa-647 and TRITC-phalloidin, respectively. A representative x/y plane is shown in each case. A deconvoluted enlargement of the pericentriolar region (boxed region) is included in the case of Cterm1-transfected cells to show that Cterm1 surrounds the microtubule-organizing center. A pile-up of 2 medial confocal stacks is presented in D-E. The staining of F-actin is presented in gray in D-F to help its visualization. A Nomarsky image of the cell field is included in all the cases. Scale bars correspond to 5 μm unless other value is indicated.$

Characterization of INF2 as a MAL-interacting protein and analysis of its subcellular distribution in Jurkat cells. (A) Yeast HF7C strain cells were cotransformed with the pAD-GAL4 255C1 cDNA obtained in the yeast 2-hybrid screen and bait constructs encoding either full-length MAL or MAL2 or the indicated D52-like prey proteins as negative controls. Yeast growth on solid media at 30°C was evaluated as follows: –, no growth after 6-8 days; ++, growth after 3-4 days; +++, growth after 1-2 days. Identical results were obtained for all 3 D52-like proteins. (B) Schematic of the INF2-1 proteins expressed in the experiments in this Figure. (C) The 1% Triton-X-100 insoluble (I) membrane fraction of Jurkat cells was separated from the soluble fraction (S) and, after solublization with 60mM octyl-glucoside, was subjected to pull-down analysis with GST alone or fused to the indicated INF2 fragments. The presence of endogenous MAL was determined by immunoblotting with mAb 6D9 (top). The Coomassie blue staining of the GST proteins used is shown (bottom). (D) Jurkat cells transiently transfected (top) or not (bottom) with Cherry-MAL were stained for endogenous INF2 using anti-INF2 antibodies and secondary antibodies coupled to Alexa-488 (top) or for INF2, α-tubulin, and F-actin using antibodies to INF2 and α-tubulin and secondary antibodies coupled to Alexa-488 or Alexa-647 and with TRITC-phalloidin, respectively (bottom). (E) Jurkat cells transiently expressing GFP fused to INF2-1 or INF2-2 were fixed and stained for α-tubulin and F-actin with anti–α-tubulin antibodies followed by secondary antibodies coupled to Alexa-647 and with TRITC-phalloidin, respectively. (F) Jurkat cells transiently expressing GFP fusions of the indicated fragments of INF2-1 were used to analyze the distribution of α-tubulin and F-actin with anti–α-tubulin antibodies followed by secondary antibodies coupled to Alexa-647 and TRITC-phalloidin, respectively. A representative x/y plane is shown in each case. A deconvoluted enlargement of the pericentriolar region (boxed region) is included in the case of Cterm1-transfected cells to show that Cterm1 surrounds the microtubule-organizing center. A pile-up of 2 medial confocal stacks is presented in D-E. The staining of F-actin is presented in gray in D-F to help its visualization. A Nomarsky image of the cell field is included in all the cases. Scale bars correspond to 5 μm unless other value is indicated.

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