3 x flag peptide  (AnaSpec)

Journal: Advanced Science

Article Title: TRAF4‐Mediated LAMTOR1 Ubiquitination Promotes mTORC1 Activation and Inhibits the Inflammation‐Induced Colorectal Cancer Progression

doi: 10.1002/advs.202301164

Figure Lengend Snippet: TRAF4 as an E3 ligase of LAMTOR1. A) Screening the E3 ubiquitin ligase of LAMTOR1. The ubiquitination of LAMTOR1 was analyzed in HEK293T cell, which was transfected with different E3, His‐Ub and LAMTOR1‐HA plasmids. The ubiquitinated proteins were pulled down under denature condition by Ni‐NTA‐agarose beads and detected via western blotting. B) The ubiquitination of LAMTOR1‐WT, but not LAMTOR1‐K151R mutant was promoted by TRAF4. C) TRAF4 promotes Lys63 linked ubiquitin chains of LAMTOR1. D) The binding of endogenous LAMTOR1 and TRAF4. Endogenous TRAF4 was immunoprecipitated from HEK293T cell by rabbit TRAF4 polyclonal antibody or control rabbit IgG antibody. Coimmunoprecipitated endogenous LAMTOR1 was analyzed via western blotting. E) His‐TRAF4 could directly interact with GST‐LAMTOR1 which was detected by GST pull‐down assay. F) TRAF4 interacted with LAMTOR1 is dependent on its C‐terminal TRAF domain. G) TRAF4 with the loss of TRAF domain could not promote LAMTOR1 ubiquitination. H) The interaction between TRAF4 and LAMTOR1 is regulated by amino acid signaling. HEK293T cell was transfected with the Flag‐TRAF4 and LAMTOR1‐HA plasmids. Then the cell was starved with amino acids deficient DMEM for 60 min and re‐stimulated with amino acids for 0, 15, 30, 60, 120 min. I) Immunofluorescent staining for endogenous TRAF4 (green) and endogenous LAMTOR1 (red). HEK293T cell was starved of amino acids for 60 min and re‐stimulated with amino acids for 5, 10, 15 min. Scale bar, 10 µm. J) Pearson's correlation quantifies endogenous TRAF4/LAMTOR1 colocalization, mean ± SD (n ≥ 5), two‐tailed Student's t test, *** p < 0.001.

Article Snippet: After 24 h, the cell lysate was collected and incubated with 15–20 μl anti‐FLAG beads for 2 h, and then Flag‐TRAF4 was eluted from beads using 3 X FLAG peptide (P9801, Beyotime).

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Mutagenesis, Binding Assay, Immunoprecipitation, Control, Pull Down Assay, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: TRAF4‐Mediated LAMTOR1 Ubiquitination Promotes mTORC1 Activation and Inhibits the Inflammation‐Induced Colorectal Cancer Progression

doi: 10.1002/advs.202301164

Figure Lengend Snippet: The ubiquitination of LAMTOR1 promotes its interaction with Rag GTPases. A,B) The interactions between LAMTOR1‐K151R and RagC or RagD were decreased comparing to LAMTOR1‐WT. Flag‐RagC or Flag‐RagD was co‐transfected with LAMTOR1‐HA WT and K151R mutant into HEK293T cell. Then the cell was lysed and RagC was immunoprecipitated by anti‐Flag antibody. Co‐immunoprecipitated LAMTOR1‐HA WT and K151R mutation were detected by anti‐HA antibody. C,D) TRAF4 promoted the interactions between LAMTOR1 and RagC or RagD. LAMTOR1‐HA and co‐precipitated RagC/D band intensities in the IP were separately performed with a normalization analysis via Image J. E) The binding of endogenous WT or K151R LAMTOR1 with RagC in HEK293T WT or KI cell lines. F) LAMTOR1 K151R mutation affects the activation of RagC using g‐amino‐hexyl‐GTP beads in HEK293T cell. GTP‐bound RagC was detected via western blotting.

Article Snippet: After 24 h, the cell lysate was collected and incubated with 15–20 μl anti‐FLAG beads for 2 h, and then Flag‐TRAF4 was eluted from beads using 3 X FLAG peptide (P9801, Beyotime).

Techniques: Ubiquitin Proteomics, Transfection, Mutagenesis, Immunoprecipitation, Binding Assay, Activation Assay, Western Blot