cells  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: The Welander TIA1 mutation dedifferentiates insulin-producing cells: Reversal by a GLP-1 receptor agonist

doi: 10.1016/j.jbc.2026.111336

Figure Lengend Snippet: The TIA1 E384 K mutation impairs MYC mRNA binding, while anti- MYC shRNA restores β-cell identity . A , WT 1 to 3 (WT, a mixture of WT clones 1–3) and Mut 1 to 3 (Mut, a mixture of Mut clones 1–3) cell levels of MYC protein were visualized by immunoblot analysis. Beta-actin was used as a loading control. The Figure shows harvested at three different occasions. B – D , WT 1 to 3 (WT, a mixture of WT clones 1–3) and Mut 1 to 3 (Mut, a mixture of Mut clones 1–3) cells, with and without treatment with palmitate (1.5 mM) and high glucose (22 mM), were RNA-immunoprecipitated using an anti-TIA1 antibody. RNA molecules binding to the TIA1 protein were isolated and quantified using RT-PCR. ( B ) MYC mRNA binding to TIA1. C , ACTN4 mRNA binding to TIA1. D , immunoblot showing immunoprecipitation of TIA1. Results are from one experiment with three replicates and are shown as means ± SEM. ∗, and ∗∗ denote p < 0.05 and 0.01, respectively, using two-way ANOVA and the Šídák post hoc test. E , WT 1 to 3 (WT, a mixture of clones 1–3) and Mut 1 to 3 (Mut, a mixture of clones 1–3) clone cells were transduced with lentiviral vectors (scramble and anti- MYC shRNA, 25 MOI). Transduction efficacy was visualized by GFP-fluorescence microscopy at 36, 60 and 84 h post transduction. Upper row (phase contrast) shows total number of cells and lower row (GFP) shows transduced cells. F , Lentivirally transduced WT 1 to 3 cells (mixture of clones 1–3) and Mut 1 to 3 cells (mixture of clones 1–3) were analyzed in three separate experiments for MYC /beta actin mRNA levels 96 h post-transduction. Results were normalized to the WT 1 to 3 scramble group. G , cells were analyzed as in F for insulin/glucagon mRNA ratios. H , cells were analyzed as in F for PAX4/ARX mRNA ratios. ∗, ∗∗∗ and ∗∗∗∗ denote p < 0.05, 0.001 and 0.0001, respectively, using one-way ANOVA and the Šídák post hoc test.

Article Snippet: After blocking 1h with 2% bovine serum albumin (BSA), the filter was incubated with the primary antibodies: rabbit anti-TIA1 (1:1000, Cell Signaling Technology), mouse anti-alpha tubulin (1:1000, Santa Cruz), mouse anti-beta-actin (1:1000, Santa Cruz), rabbit anti-c-Myc (1:1000, Cell Signaling Technology).

Techniques: Mutagenesis, Binding Assay, shRNA, Clone Assay, Western Blot, Control, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Transduction, Fluorescence, Microscopy