Journal: bioRxiv

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins

doi: 10.1101/396465

Figure Lengend Snippet: ( A-B ) HA-tagged TDP-43 (A) or Flag-tagged hnRNP A1 (B) is expressed and immunoprecipitated from HeLa cells that are treated with or without H 2 O 2 , and examined by Western blotting with indicated antibodies. hnRNP A1 shows both steady state and induced levels of PARylation, whereas no PARylation of TDP-43 was detected even when the blot is overexposed to facilitate the detection. ( C ) Recombinant purified His-tagged TDP-43 1-274 , TDP-43 274-414 and full-length hnRNP A1 proteins (4 μg) were subjected to in vitro PARylation assay and then separated on SDS-PAGE gel. Coomassie Brilliant Blue staining confirms equal protein loading (upper panel, arrows indicate the substrate proteins), followed by immunoblotting with anti-PAR (lower panel). ssDNA was added in the in vitro reaction system to activate PARP1. The PARylated bands and smears of recombinant TDP-43 and hnRNP A1 as well as PARP1 are indicated. ( D ) Functional domains of human hnRNP A1 protein. RRM: RNA recognition motif; RGG: RGG box RNA binding domain; M9: a nuclear targeting sequence termed as M9; GRD: glycine-rich domain; PBM: PAR-binding motif. The consensus sequence and the residues mutated in the PBM and putative PARylation site are indicated. h, hydrophobic amino acid residues; b, basic amino acid residues . ( E ) Confirmation of PARylation or PAR-binding deficit of K298A and PBM mut compared to WT hnRNP A1. hnRNP A1-Flag was immunoprecipitated from HeLa cells with anti-Flag and examined by Western blotting with anti-PAR. The blot is overexposed to facilitate the detection of less abundant PARylated proteins. Asterisk, the rabbit anti-HA IgG heavy chain; #, PARylated proteins co-immunoprecipitated with hnRNP A1.

Article Snippet: The supernatant was used as the soluble fraction and the pellets containing insoluble fractions were dissolved in a 9 M urea buffer (9 M urea, 50 mM Tris buffer, pH 8.0) after wash. Proteins were separated by 10% Bis-Tris SDS-PAGE (Invitrogen), immunoblotted with the primary and secondary antibodies.

Techniques: Immunoprecipitation, Western Blot, Recombinant, Purification, In Vitro, SDS Page, Staining, Functional Assay, RNA Binding Assay, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins

doi: 10.1101/396465

Figure Lengend Snippet: ( A ) Flag-tagged hnRNP A1 is expressed in HeLa cells treated with vehicle control (DMSO), Olaparib (20 μM) or H 2 O 2 (500 μM), and immunoprecipitated with anti-Flag. The immunoprecipitates are then separated by SDS-PAGE and examined by Western blotting. ( B ) The level of co-IP of TDP-43 with hnRNP A1 is quantified as ratio of co-IP of TDP-43/input TDP-43 and shown as percentage to the DMSO control group. ( C ) Western blot analysis of cells transfected with WT, K298A or PBM mut of Flag-tagged hnRNP A1, immunoprecipitated with anti-Flag, and blotted with indicated antibodies. 2xPBM mut , cells transfected with 2 times of the PBM mut expression plasmids. ( D ) The relative level of TDP-43 co-immunoprecipitated with WT or mutant hnRNP A1 is normalized to TDP-43 level in the input and shown as percentage to the WT hnRNP A1 group. Means ± SEM, n = 5 (B) or 3 (D); *p < 0.05, ** p < 0.01, *** p < 0.001; One-way ANOVA.

Article Snippet: The supernatant was used as the soluble fraction and the pellets containing insoluble fractions were dissolved in a 9 M urea buffer (9 M urea, 50 mM Tris buffer, pH 8.0) after wash. Proteins were separated by 10% Bis-Tris SDS-PAGE (Invitrogen), immunoblotted with the primary and secondary antibodies.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Transfection, Expressing, Mutagenesis