Journal: bioRxiv

Article Title: TRB3 augments IL1β-TLR4 signaling by engaging Flightless-homolog 1

doi: 10.1101/2022.10.09.511391

Figure Lengend Snippet: A) Identification of Flightless-homolog 1 as a TRB3-interacting protein using mass spectrometry. The peptides identified are highlighted in pink font. Overlapping peptides are indicated with a green arrow for start site, and blue line denoting end of the internal peptide. Only peptides with peak intensity ratio of two-fold or higher on a Log2 scale were considered. B) Co-immunoprecipitation assay examines interaction between Fli1 and TRB3 proteins using HA-tagged LRR domain of Fli1 and Flag-TRB3, co-expressed in Min6 cells, without (lane 2) and following 30 min treatment with ILβ (lane 3). HA-Fli1-LRR in the absence of TRB3 served as control (lane 1) Note: TRB3 does not interact with HA-LRR-domain of LRRFIP2 (lane 4). D) Coimmunoprecipitation assay examines interaction between Flag-TRB3 and endogenous Fli1 from Min6 cells clustered into pseudoislets, without (lane 2) and upon 30 minute treatment with ILβ (lane 3). F) Protein interaction between purified Flag-TRB3 (lanes 3, 4) incubated with lysates from human islets (lanes 2, 4) showing potential for TRB3-Fli1 in human islets. C, E , G ) Quantitative Analysis of data from Figures 2B, 2D and 2F depicted as fold induction of TRB3-Fli1 interaction over control. In each case, graphs represent average, fold-induction of TRB3-Fli interactions from three independent experiments each normalized to value of WT control ( ** p<0.005, * p<0.05) . Schematic of proximity-induced biotinylation of APEX2 (ascorbic acid-peroxidase)-TRB3 interacting proteins. Peroxidase activity of APEX2-TRB3 fusion can cleave Biotin-phenol in the presence of H 2 O 2 resulting in spontaneous biotinylation of proteins within 20 nm of TRB3. I) APEX2-TRB3 expressing Min6 cells were treated with Biotin-phenol (2 hours) followed by 1 minute of H 2 O 2 treatment. IL1β stimulation was for 30 minutes prior to peroxide treatment (lane 3). Streptavidin-agarose was used to pull down biotinylated proteins and examined for presence of Fli1 using western blotting. Biotin-phenol without peroxide treatment was used as a control (lane 1). J) Fli1 recovered in western blots of streptavidin pulldowns was quantified and presented in graph format. Again, the data are represented as average fold-difference of Fli1 recovered by streptavidin beads in three independent experiments (** p<0.005) .

Article Snippet: Plasmids encoding HA-tagged LRR (leucine-rich-repeat) domains of LRRFIP2 (Addgene plasmid # 21152), and Fli1 (Addgene plasmid # 21151), were a gift from Dr. Junying Yuan (Harvard University, Boston MA), and Flag-MyD88 (Addgene plasmid # 13093) was a gift from Dr. Ruslan Medzhitov (HHMI, Yale School of Medicine). pcDNA3-HA-Fli1 was generated by PCR-based amplification of full-length, mouse Fli1, using pCMV-Sport6 mFli1 (Horizon Discovery, Denver CO) as template, and cloned into pcDNA3.1 vector using HindIII and Xho1 sites, in frame with a C-terminal HA tag.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Purification, Incubation, Activity Assay, Expressing, Western Blot