gfp  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: Human pumilio proteins use fuzzy multivalent hydrophobic interactions to recruit the CCR4–NOT deadenylase complex to repress mRNAs

doi: 10.1016/j.jbc.2026.111281

Figure Lengend Snippet: Crosslinking mass spectrometry reveals a fuzzy binding mode of RD3:NOT module interaction . A , amino acid sequence of PUM1 RD3-RBD. RD3 is highlighted in gray and lysine (K) residues available for BS3 crosslinking are magenta . Amino acid sequences for the regions of CNOT1, CNOT2, and CNOT3 used to form the NOT module are shown below. Crosslinked residues in each subunit are bolded, with magenta lines indicating the locations of interprotein crosslinks found in the RD3–RBD:NOT module complex. B , crystal structure of the NOT module with residues that crosslink to PUM1 K794 shown with purple spheres and labeled. C , same as in ( B ) with residues that crosslink to PUM1 K718 shown with purple spheres and labeled.

Article Snippet: In co-immunoprecipitation assays, endogenous CNOT1 was detected using rabbit anti-CNOT1 (CST; catalog #: 44613) diluted 1:1000.

Techniques: Mass Spectrometry, Binding Assay, Sequencing, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Human pumilio proteins use fuzzy multivalent hydrophobic interactions to recruit the CCR4–NOT deadenylase complex to repress mRNAs

doi: 10.1016/j.jbc.2026.111281

Figure Lengend Snippet: Aliphatic and aromatic amino acid residues are essential for PUM1 RD3 activity . A , diagram of PUM1 RD3 effector protein with all IDR-associated residues L, F, and Y mutated to G (IDR muts). Position and identity of amino acid residue in the WT sequence are indicated. B , tethered function reporter assay comparing repressive activity of WT PUM1 RD3 to the IDR muts effector. N = 9 (three experimental repeats, each with three biological replicates). All data are shown as mean and individual points ± SD of log 2 fold change (log 2 FC) values relative to negative control HaloTag (HT). Significance indicated above the x-axis denotes comparisons to HT, while significance indicated below the x-axis denotes comparisons between specific effectors. For significance calling, ns = not significant where p ≥ 0.05, p < 0.0001 = ∗∗∗∗ based on ordinary one-way ANOVA and Tukey’s test for multiple comparisons. Western blot confirming the expression of V5-tagged effector proteins is shown below. Vinculin served as a loading control. C , co-immunoprecipitation of endogenous CNOT1 from HCT116 cell lysate by V5-tagged WT and IDR muts RD3. MS2-V5 served as a negative control bait protein. Vinculin served as a negative control for non-specific binding.

Article Snippet: In co-immunoprecipitation assays, endogenous CNOT1 was detected using rabbit anti-CNOT1 (CST; catalog #: 44613) diluted 1:1000.

Techniques: Activity Assay, Residue, Sequencing, Reporter Assay, Negative Control, Western Blot, Expressing, Control, Immunoprecipitation, Binding Assay