Journal: Cell systems

Article Title: Discriminatory Power of Combinatorial Antigen Recognition in Cancer T Cell Therapies

doi: 10.1016/j.cels.2020.08.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alexa647 mouse anti-myc tag clone 9B11 , Cell Signaling Technology , Cat#2233S; RRID:AB_823474.

Techniques: Recombinant, Cloning, Cell Culture, Sequencing, Software

Journal: Developmental biology

Article Title: Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

doi: 10.1016/j.ydbio.2017.08.025

Figure Lengend Snippet: Fig. 1. LBX1 is phosphorylated. (a) Myc/Flag-tagged Lbx1 were expressed in NIH3T3 cells. At 24 h post transfection, cells were lysed with lysis buffer. LBX1 was immunoprecipitated using anti-Myc antibody. Immunoprecipitates were washed and treated with shrimp alkaline phosphatase (SAP) at 37 C for 1 h, then subjected to Western blotting using anti-Flag antibody. Mobility shift of LBX1 by SAP treatment shows that LBX1 is phosphorylated in cells. (b) Seven phosphorylation sites were predicted from Scansite, as shown (green shade). The region shown by blue shade and pink shade show EH (Eps15) homology motif (a protein-protein interaction domain) and the DNA binding Homeobox domain respectively. (c) Myc-taged LBX1 and points mutants were transiently transfected to NIH3T3 cells. At 24 h post transfection, cells were lysed with lysis buffer. Wild type (Wt) LBX1 and mutants are detected by Western blotting using anti- Myc antibody and IRdye800 conjugated anti-mouse IgG. Phosphorylation of LBX1S227A was severely reduced. Phosphorylation of LBX1S223A and LBX1S234A were moderately reduced.

Article Snippet: Cells were incubated with anti-Myc mouse IgG2a monoclonal antibody (9b11, Cell Signalling Technology; 1/2000) 4 C overnight and then treated with Alexa Fluor 488 conjugated anti-mouse antibody (ab150113, Abcam; 1:400) for 30 min, followed by counterstaining with Hoechst 333258.

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Mobility Shift, Phospho-proteomics, Binding Assay

Journal: Developmental biology

Article Title: Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

doi: 10.1016/j.ydbio.2017.08.025

Figure Lengend Snippet: Fig. 2. LBX1 is phosphorylated at S223, S227, and S234. (a) Myc-taged Lbx1 and points mutants were transiently transfected to NIH3T3 cells. At 24 h post transfection, cells were treated with 100 nM PMA for 30 min and lysed. Wild type (Wt) LBX1 and mutants are detected by Western blotting using anti-Myc-tag and IRdye800 conjugated anti-mouse IgG. Phosphorylation of wild type (Wt) LBX1 was upregulated by PMA and quantitated in (b). Phosphorylation of S227A was also up- regulated by PMA treatment, suggesting LBX1 is phosphorylated at multiple sites. (c) S223 and S234 were predicted to be ERK substrates and an anti-PXSP antibody was used to detect the phosphorylation at these sites. Myc-taged Lbx1 and points mutants were transiently transfected to NIH3T3 cells. At 24 h post transfection, cells were treated with 100 nM PMA for 30 min and lysed. Wild type (Wt) LBX1 and mutants are detected by Western blotting using anti-Myc and anti-Erk substrate (PXSP) antibody. IRdye800 conjugated anti-mouse IgG and IRdye700 conjugated anti-Rabbit IgG antibodies were used as secondary antibodies. Phosphorylation at S234 but not S223 was detected by anti-PXSP antibody, revealing that S234 alone is a likely to be an ERK substrate. Mobility difference between LBX1S227A and LBX1S223A S227A double mutant reveals that S223A is also phosphorylated. Note that the phosphorylations at S223 and S227 were both increased by PMA treatment.

Article Snippet: Cells were incubated with anti-Myc mouse IgG2a monoclonal antibody (9b11, Cell Signalling Technology; 1/2000) 4 C overnight and then treated with Alexa Fluor 488 conjugated anti-mouse antibody (ab150113, Abcam; 1:400) for 30 min, followed by counterstaining with Hoechst 333258.

Techniques: Transfection, Western Blot, Phospho-proteomics, Mutagenesis