Fig. 4: Validation Screens Identify PPP1R15A (GADD34) as a Novel Regulator of IRE1α-XBP1 Signaling. | Nature Communications

Fig. 4: Validation Screens Identify PPP1R15A (GADD34) as a Novel Regulator of IRE1α-XBP1 Signaling.

From: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress

Fig. 4

A Schematic of the batch validation SPLiCR-seq screen workflow. B Heatmap showing gene knockdown phenotypes from the validation screens (top), compared to the primary screens (bottom). Genes are hierarchical clustered based on their validation screen phenotypes. Genes selected for individual validation are highlighted in blue (negative hits) and red (positive hits). C RT-PCR analysis of XBP1 splicing in HEK293T cells under Tg-induced ER stress following knockdown of PPP1R15A. Splicing levels are indicated below each lane. Three independent sgRNAs were tested. D RT-PCR analysis of XBP1 splicing in control and PPP1R15A knockdown HEK293T cells expressing either empty vector or GADD34 vector. Splicing levels are indicated below each lane. E Western blot analysis of p-IRE1α, total IRE1α, and XBP1s protein levels in control and PPP1R15A knockdown cells. GAPDH was used as a loading control. Three independent sgRNAs were tested for PPP1R15A. F RT-PCR analysis of XBP1 splicing in HEK293T cells treated with Sephin1. Splicing levels are indicated below each lane (mean ± SD, n = 3 experimental replicates). Student’s t-test: **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

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