Fig. 5

LINP1 directly interacts with eIF2α to protect eIF2α from phosphorylation at Ser51 by PERK. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the proteins interacting with LINP1 by subtracting the proteins non-specific binding to EGFP RNA after identified by HPLC–MS. “Protein processing in endoplasmic reticulum” is highlighted. B Arrows indicate the identified eIF2α peptide peak in the LINP1-pulldown sample, which was lacking in control eGFP sample. C Biotin-labeled LINP1 transcript was used to retrieve interacting protein partners by RNA pulldown with beads only and EGFP RNA as controls. The resulting protein mix from cSCC cells was applied to detect eIF2α and PERK by Western blot. D RNA immunoprecipitation (RIP) assay was performed using antibodies against eIF2α and PERK while IgG was used as control. The retrieved LINP1 RNA was detected by qPCR. U1 transcripts were used as a negative control. E Co-IP of PERK and eIF2α  followed by Western blot was performed to check whether overexpression of LINP1 influences the interaction between PERK with eIF2α. F Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to examine the co-localization of LINP1 (green) and eIF2α (red) in cSCC cells. Scale bars, 50 μm. Quantitative analysis of the fluorescence co-localization of eIF2α and LINP1 was performed. Pearson’s R value = 0.89. G Biotin-labeled full-length (FL) or domain fragments of LINP1 transcripts were used to retrieve eIF2α or PERK proteins and Western blot was performed. H In vitro phosphorylation reactions containing recombinant bacterially expressed GST–PERK was done in 50 µl kinase buffer with 0.1 mM ATP and 50 ng partially purified eIF2α. The impact of LINP1 on eIF2α phosphorylation by PERK was evaluated by adding 1 µg LINP1 transcript to the reactions. The products were analyzed by 10% SDS–PAGE. I In vitro pulldown of in vitro-expressed full-length protein, wild type NTD, mutated NTD domain containing Ser51 to Asp51 mutation and wild type CTD of eIF2α by biotinylated LINP1 transcripts were examined by Western blotting. J In vitro phosphorylation assay containing GST–PERK (20 µg), 0.1 mM ATP and 1 µg LINP1 transcript were done in 50 µl kinase buffer with 50 ng Flag-tagged in vitro-expressed full-length protein, wild type of NTD, mutated NTD domain containing Ser51 to Asp51 mutation and wild type of CTD