Flow cytometric analysis of ACHN cells, untreated (blue) or treated with UV (100 mJ/cm2, 1 hr recovery; green), using Phospho-GCN2 (Thr899) (E1V9M) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Confocal immunofluorescent analysis of HT-1376 cells (left, positive) and BT-20 cells (right, negative) using GCN2 (E7G7E) Rabbit mAb (green). Blue = DAPI #4083 (fluorescent DNA dye).
Confocal immunofluorescent analysis of HT-1376 cells, untreated (left) or treated with UV (50 mJ/cm2, 30 min recovery; right), using Phospho-GCN2 (Thr899) (E1V9M) Rabbit mAb (green). Actin filaments were labeled with Alexa Fluor® 647 Phalloidin #8940 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunoprecipitation of GCN2 protein from HT-1376 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is GCN2 (E7G7E) Rabbit mAb. Western blot analysis was performed using GCN2 (E7G7E) Rabbit mAb.
Western blot analysis of extracts from HT-1376 cells, untreated (-) or treated with UV (50 mJ/cm2, 30 min recovery; +), using Phospho-GCN2 (Thr899) (E1V9M) Rabbit mAb (upper), GCN2 (E9H6C) Rabbit mAb #40457 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from various cell lines using GCN2 (E7G7E) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.
Phosphorylation of the eukaryotic initiation factor 2 (eIF2) alpha subunit is a well-documented mechanism of downregulating protein synthesis under a variety of stress conditions. Kinases activated by viral infection (PKR), endoplasmic reticulum stress (PERK/PEK), amino acid deprivation (GCN2), and hemin deficiency (HRI) can phosphorylate the eIF2 alpha subunit (1,2). GCN2 is also required for UV light-induced translation inhibition, and in vivo phosphorylation of murine GCN2 at Thr898 is induced by both UV irradiation and by leucine deprivation (3). UV-induced activation of NF-κB also requires GCN2, which may act simply by preventing translation of IκB-alpha to replace pools that have been ubiquitinated and degraded (4). Interestingly, proteasome inhibitors (MG132 and ALLN) activate the GCN2/eIF2alpha pathway, suggesting a pivotal role for this kinase in stress response and ubiquitin-mediated signaling (5). In vitro autophosphorylation of yeast GCN2 within its activation loop at Thr882 and Thr887 (Thr898 and Thr903 in mouse) has also been reported (6).
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