Journal: Cell Death & Disease
Article Title: TIPRL potentiates survival of lung cancer by inducing autophagy through the eIF2α-ATF4 pathway
Figure Lengend Snippet: Interaction between TIPRL and eIF2α increases phosphorylation of eIF2α. a IP assays of cell lysates of A549 cells with anti-TIPRL antibody (upper panel) and cell lysates of 293 T cells with anti-HA antibody after transfection with gradual concentration of HA-tagged TIPRL (lower panel). b IP assays with anti-TIPRL antibody in A549 cells incubated in EBSS for 2 h. c Immunocytochemistry data of A549 cells where TIPRL and eIF2α proteins were double-immunostained and treated with goat anti-rabbit mouse IgG-FITC (green) and bovine anti-mouse IgG-Texas Red (red). Relative intensity of interaction were measured. d GST-pull down assay (left panel) and IP assay (right panel) with anti-HA antibody of 293 T cells co-transfected with HA-TIPRL along with GST-Mock, GST-eIF2α, GST-eIF2β, and GST-eIF2γ, respectively. e GST-pull down assay of 293T cells co-transfected with GST-eIF2α and TIPRL deletion mutants (D1–D6) (upper panel); six deletion fragments of TIPRL were sub-cloned into the pCGN-HA vector (lower panel). f IP assay of A549 cells mixed with TIPRL-mimic peptides which represent the regions of TIPRL spanning the amino acids 86–100 or 174–188. g In vitro kinase assays of phospho-eIF2α using recombinant eIF2α, gradual concentration of TIPRL, and GCN2 proteins. Phosphorylation level of eIF2α was detected by Western blots, and recombinant proteins were stained using Ponceau S (left panel). Relative intensity of phospho-eIF2α were measured (right panel). h In vitro kinase assays of phospho-eIF2α using recombinant eIF2α, GCN2, and TIPRL proteins with incubation of 100 and 200 µM of TIPRL-mimic peptides. Phosphorylation of eIF2α and transfection of peptide were detected by Western blots, and recombinant proteins were stained using Ponceau S (left panel). Relative intensity of phospho-eIF2α were measured (right panel). Depicted western blots are representatives from 2–3 independent experiment. All quantitative bar data are mean ± SEM. p -value was calculated by t -test. * P
Article Snippet: Antibody and western blotting The anti-LC3B antibody (L7543) was purchased from Sigma-Aldrich (MO, USA); HA (LF-MA0048) and GAPDH (LF-PA0212) antibodies were purchased from Ab frontier (Seoul, Republic of Korea); phospho-GCN2(T899) (ab75836), HSP60 (ab46798), Cytochrome c (ab76107), FITC (ab19224) antibodies were purchased from abcam (MA, USA); P62 (610832), HIF1α (610959) antibodies were purchased from BD bioscience (CA, USA); TIPRL antibody (a300-663a) was purchased from bethyl (TX, USA); Bip (3183), phosphor-mTOR(s2448) (5536), mTOR (2983), p-p70 s6k(T389) (9234), p-4e-bp1(T37/46) (2855), p-eIF2α(S51) (9721), eIF2α (9722), PERK (3192), MKK7 (4172), Caspase-8 (9746), Caspase-3 (9665), PARP (9542) antibodies were purchased from Cell Signaling Technology (MA, USA); β-actin (sc-47778), ATG7 (sc-33211), GCN2 (sc374609), ATF4 (sc-200), phospho-PERK(T981) (sc-32577), GST (sc-138), PP1 (sc-7482), GADD34 (sc-8327), normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027), TOM20 (sc-17764), Nbr1 (sc-130380), PP2A-Cα/β (sc-6110), Goat-anti Rabbit IgG-HRP (sc-2004), Goat-anti Mouse IgG-HRP (sc-2031), Donkey-anti Goat IgG-HRP (sc-2033) antibodies were purchased from Santa Cruz (CA, USA).
Techniques: Transfection, Concentration Assay, Incubation, Immunocytochemistry, Pull Down Assay, Clone Assay, Plasmid Preparation, In Vitro, Recombinant, Western Blot, Staining